CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/869,435, filed July 1, 2019, the disclosure of which is incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 24, 2020, is named 0133-0006W01_SL.txt and is 219,066 bytes in size.

FIELD OF THE INVENTION

[0003] This invention relates to production of immunoglobulin molecules, including methods for generating transgenic mammals capable of producing canine antigen-specific antibody-secreting cells for the generation of monoclonal antibodies.

BACKGROUND

[0004] In the following discussion certain articles and methods are described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[0005] Antibodies have emerged as important biological pharmaceuticals because they (i) exhibit exquisite binding properties that can target antigens of diverse molecular forms, (ii) are physiological molecules with desirable pharmacokinetics that make them well tolerated in treated humans and animals, and (iii) are associated with powerful immunological properties that naturally ward off infectious agents. Furthermore, established technologies exist for the rapid isolation of antibodies from laboratory animals, which can readily mount a specific antibody response against virtually any foreign substance not present natively in the body. [0006] In their most elemental form, antibodies are composed of two identical heavy (H) chains that are each paired with an identical light (L) chain. The N-termini of both H and L chains includes a variable domain (VH and VL, respectively) that together provide the paired H-L chains with a unique antigen-binding specificity.

[0007] The exons that encode the antibody VH and VL domains do not exist in the germ line DNA. Instead, each VH exon is generated by the recombination of randomly selected VH, D, and JH gene segments present in the immunoglobulin H chain locus (IGH); likewise, individual VL exons are produced by the chromosomal rearrangements of randomly selected VL and JL gene segments in a light chain locus.

[0008] The canine genome contains two alleles that can express the H chain (one allele from each parent), two alleles that can express the kappa (K) L chain, and two alleles that can express the lambda (l) L chain. There are multiple VH, D, and JH gene segments at the H chain locus as well as multiple VL and JL gene segments at both the immunoglobulin k (IGK) and immunoglobulin l (IGL) L chain loci (Collins and Watson (2018) Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire. Front. Immunol. 9:2249. (doi: 10.3389/fimmu.2018.02249)).

[0009] In a typical immunoglobulin heavy chain variable region locus, VH gene segments lie upstream (5’) of JH gene segments, with D gene segments located between the VH and JH gene segments. Downstream (3’) of the JH gene segments of the IGH locus are clusters of exons that encode the constant region (CH) of the antibody. Each cluster of CH exons encodes a different antibody class (isotype). Eight classes of antibody exist in mouse: IgM, IgD, IgG3, IgGl, IgG2a (or IgG2c), IgG2b, IgE, and IgA (at the nucleic acid level, they are respectively referred to as: m, d, g3, gΐ, y2a/c, y2b, e, and a). In canine animals (e.g., the domestic dog and wolf), the putative isotypes are IgM, IgD, IgGl, IgG2, IgG3, IgG4, IgE, and IgA (Fig. 12 A).

[00010] At the IGK locus of most mammalian species, a cluster of VK gene segments are located upstream of a small number of JK gene segments, with the JK gene segment cluster located upstream of a single CK gene. This organization of the k locus can be represented as (V _K ) _{a . . .} (JKL . . . CK, wherein a and b, independently, are an integer of 1 or more. The dog K locus is unusual in that half the VK genes are located upstream, and half are located downstream of the J _K and C _K gene segments (see schematics of the mouse IGK locus in FIG. 1C and dog IGK locus in FIG. 12C).

[00011] The IGL locus of most species includes a set of VL gene segments that are located 5’ to a variable number of J-C tandem cassettes, each made up of a T gene segment and a C gene segment (see schematic of the canine IGL locus in FIG. 12B). The organization of the l locus can be represented as (V, _. )a... (J, _. -G _. )b, wherein a and b are, independently, an integer of 1 or more. The mouse IGL locus is unusual in that it contains two units of (V )a... (K-O0 _b.

[00012] During B cell development, gene rearrangements occur first on one of the two homologous chromosomes that contain the H chain variable gene segments. The resultant VH exon is then spliced at the RNA level to the C _m exons for IgM H chain expression. Subsequently, the VL-JL rearrangements occur on one L chain allele at a time until a functional L chain is produced, after which the L chain polypeptides can associate with the IgM H chain homodimers to form a fully functional B cell receptor (BCR) for antigen. In mouse and human, as B cells continue to mature, IgD is co-expressed with IgM as alternatively spliced forms, with IgD being expressed at a level 10 times higher than IgM in the main B cell population. This contrasts with B cell development in the dog, in which the C exons are likely to be nonfunctional.

[00013] It is widely accepted by experts in the field that in mouse and human, VL-JL rearrangements first occur at the IGK locus on both chromosomes before the IGL light chain locus on either chromosome becomes receptive for VL-JL recombination. This is supported by the observation that in mouse B cells that express k light chains, the l locus on both chromosomes is usually inactivated by non-productive rearrangements. This may explain the predominant k L chain usage in mouse, which is >90% k and <10% l.

[00014] However, immunoglobulins in the dog immune system are dominated by l light chain usage, which has been estimated to be at least 90% It is not known mechanistically whether V _K -J _K rearrangements preferentially occur first over VL-JL rearrangements in canines.

[00015] Upon encountering an antigen, the B cell may undergo another round of DNA recombination at the IGH locus to remove the O _m and Cs exons, effectively switching the CH region to one of the downstream isotypes (this process is called class switching). In the dog, although cDNA clones identified as encoding canine IgGl-IgG4 have been isolated (Tang, et al. (2001) Cloning and characterization of cDNAs encoding four different canine immunoglobulin g chains. Vet. Immunol and Immunopath. 80:259 PMID 11457479), only the IgG2 constant region gene has been physically mapped to the canine IGH locus on chromosome 8 (Martin, et al. (2018) Comprehensive annotation and evolutionary insights into the canine ( Canis lupus familiaris) antigen receptor loci. Immunogenet. 70:223 doi: 10.1007/s00251-017-1028-0).

[00016] The genes encoding various canine and mouse immunoglobulins have been extensively characterized. Priat, et al., describe whole-genome radiation mapping of the dog genome in Genomics, 54:361-78 (1998), and Bao, et al., describe the molecular characterization of the VH repertoire in Canis familiaris in Veterinary Immunology and Immunopathology, 137:64-75 (2010). Martin et al. provide an annotation of the canine {Canis lupus familiaris) immunoglobulin kappa and lambda (IGK, IGL) loci, and an update to the annotation of the IGH locus in Immunogenetics, 70(4):223-236 (2018).

[00017] Blankenstein and Krawinkel describe the mouse variable heavy chain region locus in Eur. J. Immunol., 17: 1351-1357 (1987). Transgenic animals are routinely used in various research and development applications. For example, the generation of transgenic mice containing immunoglobulin genes is described in International Application WO 90/10077 and WO 90/04036. WO 90/04036 describes a transgenic mouse with an integrated human immunoglobulin "mini" locus. WO 90/10077 describes a vector containing the immunoglobulin dominant control region for use in generating transgenic animals.

[00018] Numerous methods have been developed for modifying the mouse endogenous immunoglobulin variable region gene locus with, e.g., human immunoglobulin sequences to create partly or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, e.g., U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669. However, many of the fully humanized immunoglobulin transgenic mice exhibit suboptimal antibody production because B cell development in these mice is severely hampered by inefficient V(D)J recombination, and by inability of the fully human antibodies/BCRs to function optimally with mouse signaling proteins. Other humanized immunoglobulin transgenic mice, in which the mouse coding sequences have been "swapped" with human sequences, are very time consuming and expensive to create due to the approach of replacing individual mouse exons with the syntenic human counterpart.

[00019] The use of antibodies that function as drugs is not limited to the prevention or therapy of human disease. Companion animals such as dogs suffer from some of the same afflictions as humans, e.g., cancer, atopic dermatitis and chronic pain. Monoclonal antibodies targeting IL31, CD20, IgE and Nerve Growth Factor, respectively, are already in veterinary use as for treatment of these conditions. However, before clinical use these monoclonal antibodies, which were made in mice, had to be caninized, i.e., their amino acid sequence had to be changed from mouse to dog, in order to prevent an immune response in the recipient dogs. Importantly, due to immunological tolerance, canine antibodies to canine proteins cannot be easily raised in dogs. Based on the foregoing, it is clear that a need exists for efficient and cost-effective methods to produce canine antibodies for the treatment of diseases in dogs. More particularly, there is a need in the art for small, rapidly breeding, non-canine mammals capable of producing antigen-specific canine immunoglobulins. Such non-canine mammals are useful for generating hybridomas capable of large-scale production of canine monoclonal antibodies.

[00020] PCT Publication No. 2018/189520 describes rodents and cells with a genome that is engineered to express exogenous animal immunoglobulin variable region genes from companion animals such as dogs, cats, horses, birds, rabbits, goats, reptiles, fish and amphibians.

[00021] However, there still remains a need for improved methods for generating transgenic nonhuman animals which are capable of producing an antibody with canine V regions.

SUMMARY

[00022] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[00023] Described herein is a non-canine mammalian cell and a non-canine mammal having a genome comprising an exogenously introduced partly canine immunoglobulin locus, where the introduced locus comprises coding sequences of the canine immunoglobulin variable region gene segments and non-coding sequences based on the endogenous immunoglobulin variable region locus of the non-canine mammalian host. Thus, the non canine mammalian cell or mammal is capable of expressing a chimeric B cell receptor (BCR) or antibody comprising H and L chain variable regions that are fully canine in conjunction with the respective constant regions that are native to the non-canine mammalian host cell or mammal. Preferably, the transgenic cells and animals have genomes in which part or all of the endogenous immunoglobulin variable region gene locus is removed.

[00024] At a minimum, the production of chimeric canine monoclonal antibodies in a non canine mammalian host requires the host to have at least one locus that expresses chimeric canine immunoglobulin H or L chain. In most aspects, there are one heavy chain locus and two light chain loci that, respectively, express chimeric canine immunoglobulin H and L chains.

[00025] In some aspects, the partly canine immunoglobulin locus comprises canine VH coding sequences and non-coding regulatory or scaffold sequences present in the endogenous VH gene locus of the non-canine mammalian host. In these aspects, the partly canine immunoglobulin locus further comprises canine D and JH gene segment coding sequences in conjunction with the non-coding regulatory or scaffold sequences present in the vicinity of the endogenous D and JH gene segments of the non-canine mammalian host cell genome. In one aspect, the partly canine immunoglobulin locus comprises canine VH, D and JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an endogenous immunoglobulin heavy chain locus of the non-canine mammalian host. In one aspect, the partly canine immunoglobulin locus comprises canine VH, D and JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an endogenous immunoglobulin heavy chain locus of a rodent, such as a mouse. In other aspects, the partly canine immunoglobulin locus comprises canine VL coding sequences and non-coding regulatory or scaffold sequences present in the endogenous VL gene locus of the non-canine mammalian host. In one aspect, the exogenously introduced, partly canine immunoglobulin locus comprising canine VL coding sequences further comprises canine L-chain J gene segment coding sequences and non-coding regulatory or scaffold sequences present in the vicinity of the endogenous L- chain J gene segments of the non-canine mammalian host cell genome. In one aspect, the partly canine immunoglobulin locus comprises canine V and J gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of an immunoglobulin light chain locus in the non-canine mammalian host cell. In one aspect, the partly canine immunoglobulin locus comprises canine VK and JK gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of an immunoglobulin locus of the non-canine mammalian host. In one aspect, the endogenous K locus of the non-canine mammalian host is inactivated or replaced by sequences encoding canine l chain, to increase production of canine l immunoglobulin light chain over canine K chain. In one aspect, the endogenous k locus of the non-canine mammalian host is inactivated but not replaced by sequences encoding canine l chain.

[00026] In certain aspects, the non-canine mammal is a rodent, for example, a mouse or rat.

[00027] In one aspect, the engineered immunoglobulin locus includes a partly canine immunoglobulin light chain locus that includes one or more canine l variable region gene segment coding sequences. In one aspect, the engineered immunoglobulin locus is a partly canine immunoglobulin light chain locus that includes one or more canine k variable region gene segment coding sequences.

[00028] In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising an engineered partly canine immunoglobulin locus. In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising an engineered partly canine immunoglobulin light chain locus. In one aspect, the partly canine immunoglobulin light chain locus of the rodent or rodent cell includes one or more canine immunoglobulin l variable region gene segment coding sequences. In one aspect, the partly canine immunoglobulin light chain locus of the rodent or rodent cell includes one or more canine immunoglobulin k variable region gene segment coding sequences. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising canine variable domains.

[00029] In one aspect, a transgenic rodent that produces more immunoglobulin comprising l light chain than immunoglobulin comprising k light chain is provided. In one aspect, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% l light chain immunoglobulin. In one aspect, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% l light chain immunoglobulin comprising a canine variable domain. In one aspect, more l light chain-producing cells than k light chain-producing cells are likely to be isolated from the transgenic rodent. In one aspect, more cells producing l light chain with a canine variable domain are likely to be isolated from the transgenic rodent than cells producing k light chain with a canine variable domain.

[00030] In one aspect, a transgenic rodent cell is provided that is more likely to produce immunoglobulin comprising l light chain than immunoglobulin comprising k light chain. In one aspect, the rodent cell is isolated from a transgenic rodent described herein. In one aspect, the rodent cell is recombinantly produced as described herein. In one aspect, the transgenic rodent cell or its progeny, has at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and up to about 100%, probability of producing l light chain immunoglobulin. In one aspect, the transgenic rodent cell or its progeny, has at least about about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to about 100%, probability of producing l light chain immunoglobulin with a canine variable domain

[00031] In one aspect, the engineered partly canine immunoglobulin locus comprises canine V gene segment coding sequences and Ji gene segment coding sequences and non-coding sequences such as regulatory or scaffold sequences of a rodent immunoglobulin light chain variable region gene locus.

[00032] In one aspect, the engineered immunoglobulin locus comprises canine Vi and Ji gene segment coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin l light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine Vi and Ji gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of the rodent immunoglobulin k light chain variable region gene locus. In one aspect, the partly canine immunoglobulin locus comprises one or more canine Vx gene segment coding sequences and Jx gene segment coding sequences and one or more rodent immunoglobulin l constant region coding sequences.

[00033] In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent region Cx coding sequence. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent Cx region coding and non-coding sequences. In one aspect, the rodent Cx region coding sequence is selected from a rodent C i, 0 _.2 or O _.i coding sequence. In one aspect, one or more canine Vx gene segment coding sequences are located upstream of one or more J-C units, wherein each J-C unit comprises a canine Jx gene segment coding sequence and a rodent Cx gene segment coding sequence. In one aspect, one or more canine Vx gene segment coding sequences are located upstream of one or more J-C units, wherein each J-C unit comprises a canine Jx gene segment coding sequence and a rodent Cx gene segment coding sequence and rodent Cx non-coding sequences. In one aspect, the J-C units comprise canine Jx gene segment coding sequences and rodent Cx region coding sequences embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain locus.

[00034] In one aspect, a transgenic rodent or rodent cell is provided with an engineered immunoglobulin locus that includes a rodent immunoglobulin k locus in which one or more rodent V _K gene segment coding sequences and one or more rodent J _K gene segment coding sequences have been deleted and replaced with one or more canine Vx gene segment coding sequences and one or more Jx gene segment coding sequences, respectively, and in which rodent C _K coding sequence in the locus has been replaced by rodent Cxi, Cx2, or Cx3 coding sequence(s).

[00035] In one aspect, the engineered immunoglobulin locus includes one or more canine Vx gene segment coding sequences upstream and in the same transcriptional orientation as one or more canine S, gene segment coding sequences which are upstream of one or more rodent Cx coding sequences.

[00036] In one aspect, the engineered immunoglobulin locus includes one or more canine V gene segment coding sequences upstream and in the opposite transcriptional orientation as one or more canine Ji gene segment coding sequences which are upstream of one or more rodent Cx coding sequences.

[00037] In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin k light chain locus is deleted, inactivated, or made nonfunctional by one or more of:

a. deleting or mutating all endogenous rodent V _K gene segment coding sequences; b. deleting or mutating all endogenous rodent J _K gene segment coding sequences; c. deleting or mutating endogenous rodent C _K coding sequence;

d. deleting or mutating a splice donor site, pyrimidine tract, or splice acceptor site within the intron between a J _K gene segment and C _K exon; and

e. deleting, mutating, or disrupting an endogenous intronic k enhancer (ΐEk), an 3’ enhancer sequence (3Έk), or a combination thereof.

[00038] In one aspect, a transgenic rodent or rodent cell is provided in which expression of an endogenous rodent immunoglobulin l light chain variable domain is suppressed or inactivated by one or more of:

a. deleting or mutating all endogenous rodent Vi gene segments;

b. deleting or mutating all endogenous rodent Ji gene segments;

c. deleting or mutating all endogenous rodent Cx coding sequences; and

d. deleting or mutating a splice donor site, pyrimidine tract, splice acceptor site within the intron between a Ji gene segment and C. expm, or a combination thereof.

[00039] In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine variable domain and a rodent constant domain. In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine l variable domain and rodent l constant domain. In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine k variable domain and rodent k constant domain.

[00040] In one aspect, a transgenic rodent or rodent cell is provided in which the genome of the transgenic rodent or rodent cell comprises an engineered immunoglobulin locus comprising canine V _K and J _K gene segment coding sequences. In one aspect, the canine V _K and J _K gene segment coding sequences are inserted into a rodent immunoglobulin k light chain locus. In one aspect, the canine V _K and J _K gene segment coding sequences are embedded in rodent non-coding regulatory or scaffold sequences of the rodent immunoglobulin k light chain variable region gene locus. In one aspect, the canine V _K and J _K coding sequences are inserted upstream of a rodent immunoglobulin k light chain constant region coding sequence.

[00041] In one aspect, a transgenic rodent or rodent cell is provided in which the genome of the transgenic rodent or rodent cell comprises an engineered immunoglobulin locus comprising canine V _K and J _K gene segment coding sequences inserted into a rodent immunoglobulin l light chain locus. In one aspect, the canine V _K and J _K gene segment coding sequences are embedded in rodent non-coding regulatory or scaffold sequences of the rodent immunoglobulin l light chain variable region gene locus. In one aspect, the genome of the transgenic rodent or rodent cell includes a rodent immunoglobulin k light chain constant region coding sequence inserted downstream of the canine V _K and J _K gene segment coding sequences. In one aspect, the rodent immunoglobulin k light chain constant region is inserted upstream of an endogenous rodent C, _. coding sequence. In one aspect, the rodent immunoglobulin k light chain constant region is inserted upstream of an endogenous rodent Cx coding sequence. In one aspect, expression of an endogenous rodent immunoglobulin l light chain variable domain is suppressed or inactivated by one or more of:

a. deleting or mutating all endogenous rodent V, _. gene segment coding sequences. b. deleting or mutating all endogenous rodent I _l gene segment coding sequences; c. deleting or mutating all endogenous C, _. coding sequences; and

d. deleting or mutating a splice donor site, pyrimidine tract, or splice acceptor site within the intron between a I _l gene segment and C, _. exon. [00042] In one aspect, the engineered partly canine immunoglobulin light chain locus comprises a rodent intronic k enhancer (ΐEk) and 3’ k enhancer (3Έk) regulatory sequences.

[00043] In one aspect, the transgenic rodent or rodent cell further comprises an engineered partly canine immunoglobulin heavy chain locus comprising canine immunoglobulin heavy chain variable region gene segment coding sequences and non-coding regulatory and scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the engineered canine immunoglobulin heavy chain locus comprises canine VH, D and JH gene segment coding sequences. In one aspect, each canine/rodent chimeric VH, D or JH gene segment comprises VH, D or JH coding sequence embedded in non-coding regulatory and scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the heavy chain scaffold sequences are interspersed by one or both functional ADAM6 genes.

[00044] In one aspect, the rodent regulatory and scaffold sequences comprise one or more enhancers, promoters, splice sites, introns, recombination signal sequences, or a combination thereof.

[00045] In one aspect, an endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell has been inactivated. In one aspect, an endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell has been deleted and replaced with the engineered partly canine immunoglobulin locus.

[00046] In one aspect, the rodent is a mouse or a rat. In one aspect, the rodent cell is an embryonic stem (ES) cell or a cell of an early stage embryo. In one aspect, the rodent cell is a mouse or rat embryonic stem (ES) cell, or mouse or rat cell of an early stage embryo.

[00047] In one aspect, a cell of B lymphocyte lineage is provided that is obtained from a transgenic rodent described herein, wherein the B cell expresses or is capable of expressing a chimeric immunoglobulin heavy chain or light chain comprising a canine variable region and a rodent immunoglobulin constant region. In one aspect, a hybridoma cell or immortalized cell line is provided that is derived from a cell of B lymphocyte lineage obtained from a transgenic rodent or rodent cell described herein.

[00048] In one aspect, antibodies or antigen binding portions thereof are provided that are produced by a cell from a transgenic rodent or rodent cell described herein. [00049] In one aspect, a nucleic acid sequence of a VH, D, or JH, or a YL or JL gene segment coding sequence is provided that is derived from an immunoglobulin produced by a transgenic rodent or rodent cell described herein. In one aspect, a method for generating a non-canine mammalian cell comprising a partly canine immunoglobulin locus is provided, said method comprising: a) introducing two or more recombinase targeting sites into the genome of a non-canine mammalian host cell and integrating at least one site upstream and at least one site downstream of a genomic region comprising endogenous immunoglobulin variable region genes wherein the endogenous immunoglobulin variable genes comprise VH, D and JH gene segments, or V _K and J _K gene segments, or V and J gene segments, or V , J and Cx gene segments; and b) introducing into the non-canine mammalian host cell via recombinase-mediated cassette exchange (RMCE) an engineered partly canine immunoglobulin variable gene locus comprising canine immunoglobulin variable region gene coding sequences and non-coding regulatory or scaffold sequences corresponding to the non-coding regulatory or scaffold sequences present in the endogenous immunoglobulin variable region gene locus of the non-canine mammalian host.

[00050] In another aspect, the method further comprises deleting the genomic region flanked by the two exogenously introduced recombinase targeting sites prior to step b.

[00051] In a specific aspect of this method, the exogenously introduced, engineered partly canine immunoglobulin heavy chain locus is provided that comprises canine VH gene segment coding sequences, and further comprises i) canine D and JH gene segment coding sequences and ii) non-coding regulatory or scaffold sequences upstream of the canine D gene segments (pre-D sequences, FIG. 1A) that correspond to the sequences present upstream of the endogenous D gene segments in the genome of the non-canine mammalian host. In one aspect, these upstream scaffold sequences are interspersed by non immunoglobulin genes, such as ADAM6A or ADAM6B (FIG. 1A) needed for male fertility (Nishimura et al. Developmental Biol. 233(1): 204-213 (2011)). The partly canine immunoglobulin heavy chain locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin VH gene locus and downstream of the endogenous JH gene locus on the same chromosome. In other aspects, the non-coding regulatory or scaffold sequences derive (at least partially) from other sources, e.g., they could be rationally designed artificial sequences or otherwise conserved sequences of unknown functions, sequences that are a combination of canine and artificial or other designed sequences, or sequences from other species. As used herein,“artificial sequence” refers to a sequence of a nucleic acid not derived from a sequence naturally occurring at a genetic locus. In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain variable region locus. In another aspect, the non coding regulatory or scaffold sequences are rodent immunoglobulin heavy chain variable region non-coding or scaffold sequences.

[00052] In yet another specific aspect of the method, the introduced engineered partly canine immunoglobulin locus comprises canine immunoglobulin VL gene segment coding sequences, and further comprises i) canine L-chain J gene segment coding sequences and ii) non-coding regulatory or scaffold sequences corresponding to the non-coding regulatory or scaffold sequences present in the endogenous L chain locus of the non-canine mammalian host cell genome. In one aspect, the engineered partly canine immunoglobulin locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin VL gene locus and downstream of the endogenous J gene locus on the same chromosome.

[00053] In a more particular aspect of this method, an exogenously introduced, engineered partly canine immunoglobulin light chain locus is provided that comprises canine V gene segment coding sequences and canine J gene segment coding sequences. In one aspect, the partly canine immunoglobulin light chain locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin V gene locus and downstream of the endogenous k gene locus on the same chromosome.

[00054] In one aspect, the exogenously introduced, engineered partly canine immunoglobulin light chain locus comprises canine V _K gene segment coding sequences and canine J _K gene segment coding sequences. In one aspect, the partly canine immunoglobulin light chain locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin V _K gene locus and downstream of the endogenous J _K gene locus on the same chromosome.

[00055] In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent l immunoglobulin light chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin l light chain variable region locus. In another aspect, the non-coding regulatory or scaffold sequences are rodent immunoglobulin l light chain variable region non-coding or scaffold sequences.

[00056] In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain variable region locus. In another aspect, the non-coding regulatory or scaffold sequences are rodent immunoglobulin k light chain variable region non-coding or scaffold sequences.

[00057] In one aspect, the engineered partly canine immunoglobulin locus is synthesized as a single nucleic acid, and introduced into the non-canine mammalian host cell as a single nucleic acid region. In one aspect, the engineered partly canine immunoglobulin locus is synthesized in two or more contiguous segments, and introduced to the mammalian host cell as discrete segments. In another aspect, the engineered partly canine immunoglobulin locus is produced using recombinant methods and isolated prior to being introduced into the non-canine mammalian host cell.

[00058] In another aspect, methods for generating a non-canine mammalian cell comprising an engineered partly canine immunoglobulin locus are provided, said method comprising: a) introducing into the genome of a non-canine mammalian host cell two or more sequence- specific recombination sites that are not capable of recombining with one another, wherein at least one recombination site is introduced upstream of an endogenous immunoglobulin variable region gene locus while at least one recombination site is introduced downstream of the endogenous immunoglobulin variable region gene locus on the same chromosome; b) providing a vector comprising an engineered partly canine immunoglobulin locus having i) canine immunoglobulin variable region gene coding sequences and ii) non-coding regulatory or scaffold sequences based on an endogenous immunoglobulin variable region gene locus of the host cell genome, wherein the partly canine immunoglobulin locus is flanked by the same two sequence-specific recombination sites that flank the endogenous immunoglobulin variable region gene locus of the host cell of a); c) introducing into the host cell the vector of step b) and a site specific recombinase capable of recognizing the two recombinase sites; d) allowing a recombination event to occur between the genome of the cell of a) and the engineered partly canine immunoglobulin locus, resulting in a replacement of the endogenous immunoglobulin variable region gene locus with the engineered partly canine immunoglobulin variable region gene locus.

[00059] In one aspect, the partly canine immunoglobulin locus comprises VH immunoglobulin gene segment coding sequences, and further comprises i) canine D and JH gene segment coding sequences, ii) non-coding regulatory or scaffold sequences surrounding the codons of individual VH, D, and JH gene segments present endogenously in the genome of the non-canine mammalian host, and iii) pre-D sequences based on the endogenous genome of the non-canine mammalian host cell. The recombinase targeting sites are introduced upstream of the endogenous immunoglobulin VH gene locus and downstream of the endogenous D and JH gene locus.

[00060] In one aspect, there is provided a transgenic rodent with a genome deleted of a rodent endogenous immunoglobulin variable gene locus and in which the deleted rodent endogenous immunoglobulin variable gene locus has been replaced with an engineered partly canine immunoglobulin locus comprising canine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the rodent endogenous immunoglobulin variable gene locus, wherein the engineered partly canine immunoglobulin locus of the transgenic rodent is functional and expresses immunoglobulin chains with canine variable domains and rodent constant domains. In some aspects, the engineered partly canine immunoglobulin locus comprises canine VH, D, and JH coding sequences, and in some aspects, the engineered partly canine immunoglobulin locus comprises canine VL and JL coding sequences. In one aspect, the partly canine immunoglobulin locus comprises canine V and J coding sequences. In another aspect, the partly canine immunoglobulin locus comprises canine V _K and J _K coding sequences.

[00061] Some aspects provide a cell of B lymphocyte lineage from the transgenic rodent, a part or whole immunoglobulin molecule comprising canine variable domains and rodent constant domains obtained from the cell of B lymphocyte lineage, a hybridoma cell derived from the cell of B lymphocyte lineage, a part or whole immunoglobulin molecule comprising canine variable domains and rodent constant domains obtained from the hybridoma cell, a part or whole immunoglobulin molecule comprising canine variable domains derived from an immunoglobulin molecule obtained from the hybridoma cell, an immortalized cell derived from the cell of B lymphocyte lineage, a part or whole immunoglobulin molecule comprising canine variable domains and rodent constant domains obtained from the immortalized cell, a part or whole immunoglobulin molecule comprising canine variable domains derived from an immunoglobulin molecule obtained from the immortalized cell.

[00062] In one aspect, a transgenic rodent is provided, wherein the engineered partly canine immunoglobulin locus comprises canine VL and JL coding sequences, and a transgenic rodent, wherein the engineered partly canine immunoglobulin loci comprise canine VH, D, and JH or VL and JL coding sequences. In some aspects, the rodent is a mouse. In some aspects, the non-coding regulatory sequences comprise the following sequences of endogenous host origin: promoters preceding each V gene segment coding sequence, introns, splice sites, and recombination signal sequences for V(D)J recombination; in other aspects, the engineered partly canine immunoglobulin locus further comprises one or more of the following sequences of endogenous host origin: ADAM6A or ADAM6B gene, a Pax-5-Activated Intergenic Repeat (PAIR) elements, or CTCF binding sites from a heavy chain intergenic control region 1.

[00063] In one aspect, the non-canine mammalian cell for use in each of the above methods is a mammalian cell, for example, a mammalian embryonic stem (ES) cell. In one aspect, the mammalian cell is a cell of an early stage embryo. In one aspect, the non-canine mammalian cell is a rodent cell. In one aspect, the non-canine mammalian cell is a mouse cell. [00064] Once the cells have been subjected to the replacement of the endogenous immunoglobulin variable region gene locus by the introduced partly canine immunoglobulin variable region gene locus, the cells can be selected and isolated. In one aspect, the cells are non-canine mammalian ES cells, for example, rodent ES cells, and at least one isolated ES cell clone is then utilized to create a transgenic non-canine mammal expressing the engineered partly canine immunoglobulin variable region gene locus.

[00065] In one aspect, a method for generating the transgenic rodent is provided, said method comprising: a) integrating at least one target site for a site-specific recombinase in a rodent cell’s genome upstream of an endogenous immunoglobulin variable gene locus and at least one target site for a site-specific recombinase downstream of the endogenous immunoglobulin variable gene locus, wherein the endogenous immunoglobulin variable locus comprises VH, D and JH gene segments, or V _K and J _K gene segments, or V and J gene segments, or V , E and C. gene segments; b) providing a vector comprising an engineered partly canine immunoglobulin locus, said engineered partly canine immunoglobulin locus comprising chimeric canine immunoglobulin gene segments, wherein each of the partly canine immunoglobulin gene segment comprises canine immunoglobulin variable gene coding sequences and rodent non-coding regulatory or scaffold sequences, with the partly canine immunoglobulin variable gene locus being flanked by target sites for a site-specific recombinase wherein the target sites are capable of recombining with the target sites introduced into the rodent cell; c) introducing into the cell the vector and a site-specific recombinase capable of recognizing the target sites; d) allowing a recombination event to occur between the genome of the cell and the engineered partly canine immunoglobulin locus resulting in a replacement of the endogenous immunoglobulin variable gene locus with the engineered partly canine immunoglobulin locus; e) selecting a cell that comprises the engineered partly canine immunoglobulin variable locus generated in step d); and utilizing the cell to create a transgenic rodent comprising partly canine the engineered partly canine immunoglobulin variable locus. In some aspects, the cell is a rodent embryonic stem (ES) cell, and in some aspects the cell is a mouse embryonic stem (ES) cell. Some aspects of this method further comprise after, after step a) and before step b), a step of deleting the endogenous immunoglobulin variable gene locus by introduction of a recombinase that recognizes a first set of target sites, wherein the deleting step leaves in place at least one set of target sites that are not capable of recombining with one another in the rodent cell’s genome. In some aspects, the vector comprises canine VH, D, and JH, coding sequences, and in some aspects the vector comprises canine VL and JL coding sequences. In some aspects, the vector further comprises rodent promoters, introns, splice sites, and recombination signal sequences of variable region gene segments.

[00066] In another aspect, a method for generating a transgenic non-canine mammal comprising an exogenously introduced, engineered partly canine immunoglobulin variable region gene locus is provided, said method comprising: a) introducing into the genome of a non-canine mammalian host cell one or more sequence-specific recombination sites that flank an endogenous immunoglobulin variable region gene locus and are not capable of recombining with one another; b) providing a vector comprising a partly canine immunoglobulin locus having i) canine variable region gene coding sequences and ii) non coding regulatory or scaffold sequences based on the endogenous host immunoglobulin variable region gene locus, wherein the coding and non-coding regulatory or scaffold sequences are flanked by the same sequence-specific recombination sites as those introduced to the genome of the host cell of a); c) introducing into the cell the vector of step b) and a site-specific recombinase capable of recognizing one set of recombinase sites; d) allowing a recombination event to occur between the genome of the cell of a) and the engineered partly canine immunoglobulin variable region gene locus, resulting in a replacement of the endogenous immunoglobulin variable region gene locus with the partly canine immunoglobulin locus; e) selecting a cell which comprises the partly canine immunoglobulin locus; and f) utilizing the cell to create a transgenic animal comprising the partly canine immunoglobulin locus.

[00067] In a specific aspect, the engineered partly canine immunoglobulin locus comprises canine VH, D, and JH gene segment coding sequences, and non-coding regulatory and scaffold pre-D sequences (including a fertility-enabling gene) present in the endogenous genome of the non-canine mammalian host. In one aspect, the sequence-specific recombination sites are then introduced upstream of the endogenous immunoglobulin VH gene segments and downstream of the endogenous JH gene segments. [00068] In one aspect, a method for generating a transgenic non-canine animal comprising an engineered partly canine immunoglobulin locus is provided, said method comprising: a) providing a non-canine mammalian cell having a genome that comprises two sets of sequence-specific recombination sites that are not capable of recombining with one another, and which flank a portion of an endogenous immunoglobulin variable region gene locus of the host genome; b) deleting the portion of the endogenous immunoglobulin locus of the host genome by introduction of a recombinase that recognizes a first set of sequence- specific recombination sites, wherein such deletion in the genome retains a second set of sequence-specific recombination sites; c) providing a vector comprising an engineered partly canine immunoglobulin variable region gene locus having canine coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene locus, where the coding and non-coding regulatory or scaffold sequences are flanked by the second set of sequence-specific recombination sites; d) introducing the vector of step c) and a site-specific recombinase capable of recognizing the second set of sequence-specific recombination sites into the cell; e) allowing a recombination event to occur between the genome of the cell and the partly canine immunoglobulin locus, resulting in a replacement of the endogenous immunoglobulin locus with the engineered partly canine immunoglobulin variable locus; f) selecting a cell that comprises the partly canine immunoglobulin variable region gene locus; and g) utilizing the cell to create a transgenic animal comprising the engineered partly canine immunoglobulin variable region gene locus.

[00069] In one aspect, a method for generating a transgenic non-canine mammal comprising an engineered partly canine immunoglobulin locus is provided, said method comprising: a) providing a non-canine mammalian embryonic stem ES cell having a genome that contains two sequence-specific recombination sites that are not capable of recombining with each other, and which flank the endogenous immunoglobulin variable region gene locus; b) providing a vector comprising an engineered partly canine immunoglobulin locus comprising canine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene locus, where the partly canine immunoglobulin locus is flanked by the same two sequence-specific recombination sites that flank the endogenous immunoglobulin variable region gene locus in the ES cell; c) bringing the ES cell and the vector into contact with a site-specific recombinase capable of recognizing the two recombinase sites under appropriate conditions to promote a recombination event resulting in the replacement of the endogenous immunoglobulin variable region gene locus with the engineered partly canine immunoglobulin variable region gene locus in the ES cell; d) selecting an ES cell that comprises the engineered partly canine immunoglobulin locus; and e) utilizing the cell to create a transgenic animal comprising the engineered partly canine immunoglobulin locus.

[00070] In one aspect, the transgenic non-canine mammal is a rodent, e.g., a mouse or a rat.

[00071] In one aspect, a non-canine mammalian cell and a non-canine transgenic mammal are provide that express an introduced immunoglobulin variable region gene locus having canine variable region gene coding sequences and non-coding regulatory or scaffold sequences based on the endogenous non-canine immunoglobulin locus of the host genome, where the non-canine mammalian cell and transgenic animal express chimeric antibodies with fully canine H or L chain variable domains in conjunction with their respective constant regions that are native to the non-canine mammalian cell or animal.

[00072] Further, B cells from transgenic animals are provided that are capable of expressing partly canine antibodies having fully canine variable sequences, wherein such B cells are immortalized to provide a source of a monoclonal antibody specific for a particular antigen. In one aspect, a cell of B lymphocyte lineage from a transgenic animal is provided that is capable of expressing partly canine heavy or light chain antibodies comprising a canine variable region and a rodent constant region.

[00073] In one aspect, canine immunoglobulin variable region gene sequences cloned from B cells are provided for use in the production or optimization of antibodies for diagnostic, preventative and therapeutic uses.

[00074] In one aspect, hybridoma cells that are are provided that are capable of producing partly canine monoclonal antibodies having fully canine immunoglobulin variable region sequences. In one aspect, a hybridoma or immortalized cell line of B lymphocyte lineage is provided.

[00075] In another aspect, antibodies or antigen binding portions thereof produced by a transgenic animal or cell described herein are provided. In another aspect, antibodies or antigen binding portions thereof comprising variable heavy chain or variable light chain sequences derived from antibodies produced by a transgenic animal or cell described herein are provided.

[00076] In one aspect, methods for determining the sequences of the H and L chain immunoglobulin variable domains from the monoclonal antibody-producing hybridomas or primary plasma cells or B cells and combining the VH and VL sequences with canine constant regions are provided for creating a fully canine antibody that is not immunogenic when injected into dogs.

[00077] These and other aspects, objects and features are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

[00078] FIG. 1 A is a schematic diagram of the endogenous mouse IGH locus located at the telomeric end of chromosome 12.

[00079] FIG. IB is a schematic diagram of the endogenous mouse IGL locus located on chromosome 16.

[00080] FIG. 1C is a schematic diagram of the endogenous mouse IGK locus located on chromosome 6.

[00081] FIG. 2 is a schematic diagram illustrating the strategy of targeting by homologous recombination to introduce a first set of sequence-specific recombination sites into a region upstream of the H chain variable region gene locus in the genome of a non-canine mammalian host cell.

[00082] FIG. 3 is another schematic diagram illustrating the strategy of targeting by homologous recombination to introduce a first set of sequence-specific recombination sites into a region upstream of the H chain variable region gene locus in the genome of a non canine mammalian host cell.

[00083] FIG. 4 is a schematic diagram illustrating the introduction of a second set of sequence-specific recombination sites into a region downstream of the H chain variable region gene locus in the genome of a non-canine mammalian cell via a homology targeting vector. [00084] FIG. 5 is a schematic diagram illustrating deletion of the endogenous immunoglobulin H chain variable region gene locus from the genome of the non-canine mammalian host cell.

[00085] FIG. 6 is a schematic diagram illustrating the RMCE strategy to introduce an engineered partly canine immunoglobulin H chain locus into the non-canine mammalian host cell genome that has been previously modified to delete the endogenous immunoglobulin H chain variable region gene locus.

[00086] FIG. 7 is a schematic diagram illustrating the RMCE strategy to introduce an engineered partly canine immunoglobulin H chain locus comprising additional regulatory sequences into the non-canine mammalian host cell genome that has been previously modified to delete the endogenous immunoglobulin H chain variable region genes.

[00087] FIG. 8 is a schematic diagram illustrating the introduction of an engineered partly canine immunoglobulin H chain variable region gene locus into the endogenous immunoglobulin H chain locus of the mouse genome.

[00088] FIG. 9 is a schematic diagram illustrating the introduction of an engineered partly canine immunoglobulin k L chain variable region gene locus into the endogenous immunoglobulin k L chain locus of the mouse genome.

[00089] FIG. 10 is a schematic diagram illustrating the introduction of an engineered partly canine immunoglobulin l L chain variable region gene locus into the endogenous immunoglobulin l L chain locus of the mouse genome.

[00090] FIG. 11 is a schematic diagram illustrating the introduction of an engineered partly canine immunoglobulin locus comprising a canine VH minilocus via RMCE.

[00091] FIG. 12A is a schematic diagram of the endogenous canine IGH locus located on chromosome 8 showing the entire Igh locus (1201) and an expanded view of the IGHC region (1202).

[00092] FIG. 12B is a schematic diagram of the endogenous canine IGL locus located on chromosome 26.

[00093] FIG. 12C is a schematic diagram of the endogenous canine IGK locus located on chromosome 17. Arrows indicate the transcriptional orientation of the V _K gene segments. In the native canine IGK locus (1220) some V _K gene segments are downstream of the C _K exon. In the partly canine Ig _K locus described herein (1221), all of the V _K gene segment coding sequences are upstream of the C _K exon and in the same transcriptional orientation as the CK exon (See Example 4).

[00094] FIG. 13 is a schematic diagram illustrating an engineered partly canine immunoglobulin light chain variable region locus in which one or more canine V, _. gene segment coding sequences are inserted into a rodent immunoglobulin k light chain locus upstream of one or more canine E gene segment coding sequences, which are upstream of one or more rodent Cx region coding sequences.

[00095] FIG. 14 is a schematic diagram illustrating the introduction of an engineered partly canine light chain variable region locus in which one or more canine V, _. gene segment coding sequences are inserted into a rodent immunoglobulin k light chain locus upstream of an array of E-Cx tandem cassettes in which the E is of canine origin and the C, _. is of mouse origin, C i, Cx2 or Cx3.

[00096] FIG. 15 shows flow cytometry profiles of 293 T/l 7 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV3-5-mouse O _m membrane form IgM ^b allotype, and canine IGLV3-28/E6 attached to various combinations of mouse C _K and Cx (1501), or canine IGKV2-5/E1 attached to various combinations of mouse C _K and Cx (1502). The cell s have been stained for cell surface hCD4 ( 1509) or for mouse IgM ^b (1510).

[00097] FIG.16 shows flow cytometry profiles of 293T/17 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV3-5-mouse O _m membrane form IgM ^b allotype, and canine IGLV3-28/E6 attached to various combinations of mouse C _K and Cx (1601), or canine IGKV2-5/E1 attached to various combinations of mouse C _K and Cx (1602). The cells have been stained for cell surface mouse ELC (1601) or mouse KLC (1602).

[00098] FIG. 17 shows flow cytometry profiles of 293T/17 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV4-1 -mouse C _m membrane form IgM ^b allotype, and canine IGLV3-28/E6 attached to various combinations of mouse C _K and Cx (1701), or canine IGKV2-5/E1 attached to various combinations of mouse C _K and Cx (1702). The cells have been stained for cell surface hCD4 (1709) or for mouse IgM ^b (1710).

[00099] FIG. 18 shows flow cytometry profiles of 293 T/l 7 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV3- 19-mouse C _m membrane form IgM ^b allotype, and canine IGLV3-28/Jx6 attached to various combinations of mouse C _K and Cx (1801), or canine IGKV2-5/J _K l attached to various combinations of mouse C _K and Cx (1802). The cells have been stained for cell surface hCD4 (1809) or for mouse IgM ^b (1810).

[000100] FIG. 19A shows western blots of culture supernatants and FIG. 19B shows western blots of cell lysates of 393T/17 cells transfected with expression vectors encoding canine IGHV3-5 attached to mouse C _Y 2 _a (1901), IGHV3-19 attached to mouse C _Y 2 _a (1902) or IGHV4-1 attached to mouse C _Y 2 _« (1903) and canine IGLV3-28/L6 attached to various combinations of mouse C _K (1907) and Cx (1908-1910). The samples were electrophoresed under reducing conditions and the blot was probed with an anti-mouse IgG2a antibody.

[000101] FIG. 20A shows western blot loading control Myc for the cell lysates from FIG. 18 and FIG. 20B shows western blot loading control GAPDH for the cell lysates from FIG. 18.

[000102] FIG. 21A shows western blots of culture supernatants (non-reducing conditions) and FIG. 21B shows western blots of cell lysates (reducing conditions) of 393T/17 cells transfected with expression vectors encoding canine IGHV3-5-mouse C _Y 2 _a and canine IGLV3-28/Jx6 attached to various combinations of mouse C _K (2102) and Cx (2103, 2104) or transfected with expression vectors encoding canine IGHV3-5-mouse C _Y 2 _a and canine IGKV2-5/J _k 1 attached to various combinations of mouse C _K (2105) and Cx (2106, 2107). The blots in FIG. 21 A were probed with antibodies to mouse IgG2a and the blots in FIG. 2 IB were probed with antibodies to mouse k LC.

[000103] FIG. 22 shows flow cytometry profiles of 293T/17 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV3-5 attached to mouse Cs membrane form, and canine IGKV2-5/J _K l attached to mouse C _K (2201) or canine IGLV3-28/L6 attached to mouse C i, Cx2 or Cx ₃ (2202-2204). The cells have been stained for cell surface hCD4 (2205), mouse CD79b (2206), mouse IgD (2207), mouse k LC (2208), or mouse l LC (2209).

[000104] FIG. 23 shows flow cytometry profiles of 293T/17 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV3-19 attached to mouse Cs membrane form, and canine IGKV2-5/J _K l attached to mouse C _K (2301) or canine IGLV3-28/L6 attached to mouse Cxi, Cx2 or Cx ₃ (2302-2304). The cells have been stained for cell surface hCD4 (2205), mouse CD79b (2206), mouse IgD (2207), mouse k LC (2208), or mouse l LC (2209).

[000105] FIG. 24 shows flow cytometry profiles of 293T/17 cells transfected with expression vectors encoding human CD4 (hCD4), canine IGHV4-1 attached to mouse Cs membrane form, and canine IGKV2-5/J _K l attached to mouse C _K (2401) or canine IGLV3-28/L6 attached to mouse C i, Cx2 or Cx3 (2402-2404). The cells have been stained for cell surface hCD4 (2405), mouse CD79b (2406), mouse IgD (2407), mouse k LC (2408), or mouse l LC (2409).

DEFINITIONS

[000106] The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.

[000107] The term“locus” as used herein refers to a chromosomal segment or nucleic acid sequence that, respectively, is present endogenously in the genome or is (or about to be) exogenously introduced into the genome. For example, an immunoglobulin locus may include part or all of the genes (i.e., V, D, J gene segments as well as constant region genes) and intervening sequences (i.e., introns, enhancers, etc.) supporting the expression of immunoglobulin H or L chain polypeptides. Thus, a locus (e.g., immunoglobulin heavy chain variable region gene locus) may refer to a specific portion of a larger locus (e.g., a portion of the immunoglobulin H chain locus that includes the VH, DH and JH gene segments). Similarly, an immunoglobulin light chain variable region gene locus may refer to a specific portion of a larger locus (e.g., a portion of the immunoglobulin L chain locus that includes the VL and JL gene segments). The term "immunoglobulin variable region gene" as used herein refers to a V, D, or J gene segment that encodes a portion of an immunoglobulin H or L chain variable domain. The term“immunoglobulin variable region gene locus” as used herein refers to part of, or the entire, chromosomal segment or nucleic acid strand containing clusters of the V, D, or J gene segments and may include the non coding regulatory or scaffold sequences. [000108] The term“gene segment” as used herein, refers to a nucleic acid sequence that encodes a part of the heavy chain or light chain variable domain of an immunoglobulin molecule. A gene segment can include coding and non-coding sequences. The coding sequence of a gene segment is a nucleic acid sequence that can be translated into a polypeptide, such the leader peptide and the N-terminal portion of a heavy chain or light chain variable domain. The non-coding sequences of a gene segment are sequences flanking the coding sequence, which may include the promoter, 5’ untranslated sequence, intron intervening the coding sequences of the leader peptide, recombination signal sequence(s) (RSS), and splice sites. The gene segments in the immunoglobulin heavy chain (IGH) locus comprise the VH, D and JH gene segments (also referred to as IGHV, IGHD and IGHJ, respectively). The light chain variable region gene segments in the immunoglobulin k and l light loci can be referred to as VL and JL gene segments. In the k light chain, the VL and JL gene segments can be referred to as V _K and J _K gene segments or IGKV and IGKJ. Similarly, in the l light chain, the VL and JL gene segments can be referred to as V and JL gene segments or IGLV and IGLJ.

[000109] The heavy chain constant region can be referred to as CH or IGHC. The CH region exons that encode IgM, IgD, IgGl-4, IgE, or IgA can be referred to as, respectively, O _m , Ca, C _7I -4, Ce or C _a. Similarly, the immunoglobulin k or l constant region can be referred to as C _K or C _L , as well as IGKC or IGLC, respectively.

[000110] "Partly canine" as used herein refers to a strand of nucleic acids, or their expressed protein and RNA products, comprising sequences corresponding to the sequences found in a given locus of both a canine and a non-canine mammalian host. "Partly canine" as used herein also refers to an animal comprising nucleic acid sequences from both a canine and a non-canine mammal, for example, a rodent. In one aspect, the partly canine nucleic acids have coding sequences of canine immunoglobulin H or L chain variable region gene segments and sequences based on the non-coding regulatory or scaffold sequences of the endogenous immunoglobulin locus of the non-canine mammal.

[000111] The term "based on" when used with reference to endogenous non-coding regulatory or scaffold sequences from a non-canine mammalian host cell genome refers to the non-coding regulatory or scaffold sequences that are present in the corresponding endogenous locus of the mammalian host cell genome. In one aspect, the term“based on” means that the non-coding regulatory or scaffold sequences that are present in the partly canine immunoglobulin locus share a relatively high degree of homology with the non coding regulatory or scaffold sequences of the endogenous locus of the host mammal. In one aspect, the non-coding sequences in the partly canine immunoglobulin locus share at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology with the corresponding non-coding sequences found in the endogenous locus of the host mammal. In one aspect, the non-coding sequences in the partly canine immunoglobulin locus are retained from an immunoglobulin locus of the host mammal. In one aspect, the canine coding sequences are embedded in the non-regulatory or scaffold sequences of the immunoglobulin locus of the host mammal. In one aspect, the host mammal is a rodent, such as a rat or mouse.

[000112] “Non-coding regulatory sequences” refer to sequences that are known to be essential for (i) V(D)J recombination, (ii) isotype switching, (iii) proper expression of the full-length immunoglobulin H or L chains following V(D)J recombination, and (iv) alternate splicing to generate, e.g., membrane and secreted forms of the immunoglobulin H chain.“Non-coding regulatory sequences” may further include the following sequences of endogenous origin: enhancer and locus control elements such as the CTCF and PAIR sequences (Proudhon, et al., Adv. Immunol. 128: 123-182 (2015)); promoters preceding each endogenous V gene segment; splice sites; introns; recombination signal sequences flanking each V, D, or J gene segment. In one aspect, the“non-coding regulatory sequences” of the partly canine immunoglobulin locus share at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and up to 100% homology with the corresponding non-coding sequences found in the targeted endogenous immunoglobulin locus of the non canine mammalian host cell.

[000113] “Scaffold sequences” refer to sequences intervening the gene segments present in the endogenous immunoglobulin locus of the host cell genome. In certain aspects, the scaffold sequences are interspersed by sequences essential for the expression of a functional non-immunoglobulin gene, for example, ADAM6A or ADAM6B. In certain aspects, the scaffold sequences are derived (at least partially) from other sources— e.g., they could be rationally designed or artificial sequences, sequences present in the immunoglobulin locus of the canine genome, sequences present in the immunoglobulin locus of another species, or a combination thereof. It is to be understood that the phrase “non-coding regulatory or scaffold sequence” is inclusive in meaning (i.e., referring to both the non-coding regulatory sequence and the scaffold sequence existing in a given locus).

[000114] The term "homology targeting vector" refers to a nucleic acid sequence used to modify the endogenous genome of a mammalian host cell by homologous recombination; such nucleic acid sequence may comprise (i) targeting sequences with significant homologies to the corresponding endogenous sequences flanking a locus to be modified that is present in the genome of the non-canine mammalian host, (ii) at least one sequence- specific recombination site, (iii) non-coding regulatory or scaffold sequences, and (iv) optionally one or more selectable marker genes. As such, a homology targeting vector can be used to introduce a sequence-specific recombination site into particular region of a host cell genome.

[000115] "Site-specific recombination" or“sequence-specific recombination” refers to a process of DNA rearrangement between two compatible recombination sequences (also referred to as“sequence-specific recombination sites” or“site-specific recombination sequences”) including any of the following three events: a) deletion of a preselected nucleic acid flanked by the recombination sites; b) inversion of the nucleotide sequence of a preselected nucleic acid flanked by the recombination sites, and c) reciprocal exchange of nucleic acid sequences proximate to recombination sites located on different nucleic acid strands. It is to be understood that this reciprocal exchange of nucleic acid segments can be exploited as a targeting strategy to introduce an exogenous nucleic acid sequence into the genome of a host cell.

[000116] The term "targeting sequence" refers to a sequence homologous to DNA sequences in the genome of a cell that flank or are adjacent to the region of an immunoglobulin locus to be modified. The flanking or adjacent sequence may be within the locus itself or upstream or downstream of coding sequences in the genome of the host cell. Targeting sequences are inserted into recombinant DNA vectors which are used to transfect, e.g., ES cells, such that sequences to be inserted into the host cell genome, such as the sequence of a recombination site, are flanked by the targeting sequences of the vector.

[000117] The term "site-specific targeting vector" as used herein refers to a vector comprising a nucleic acid encoding a sequence-specific recombination site, an engineered partly canine locus, and optionally a selectable marker gene, which is used to modify an endogenous immunoglobulin locus in a host using recombinase-mediated site-specific recombination. The recombination site of the targeting vector is suitable for site-specific recombination with another corresponding recombination site that has been inserted into a genomic sequence of the host cell (e.g., via a homology targeting vector), adjacent to an immunoglobulin locus that is to be modified. Integration of an engineered partly canine sequence into a recombination site in an immunoglobulin locus results in replacement of the endogenous locus by the exogenously introduced partly canine region.

[000118] The term "transgene" is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a cell, and particularly a cell of a mammalian host animal. The term "transgene" as used herein refers to a partly canine nucleic acid, e.g., a partly canine nucleic acid in the form of an engineered expression construct or a targeting vector.

[000119] "Transgenic animal" refers to a non-canine animal, usually a mammal, having an exogenous nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). In one aspect, a partly canine nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to methods well known in the art.

[000120] A "vector" includes plasmids and viruses and any DNA or RNA molecule, whether self-replicating or not, which can be used to transform or transfect a cell.

[000121] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a locus" refers to one or more loci, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[000122] As used herein, the term "or" can mean "and/or", unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive. The terms "including," "includes" and "included", are not limiting.

[000123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.

[000124] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[000125] The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, polymerase chain reaction, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach and Veksler, Eds. (2007), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3.sup.rd Ed., W. H. Freeman Pub., New York, N.Y.; and Berg et al. (2002) Biochemistry, 5.sup.th Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

DETAILED DESCRIPTION

[000126] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[000127] Described herein is a transgenic rodent or rodent cell having a genome comprising an engineered partly canine immunoglobulin heavy chain or light chain locus. In one aspect, the partly canine immunoglobulin heavy chain locus comprises one or more canine immunoglobulin heavy chain variable region gene segments. In one aspect, the partly canine immunoglobulin light chain locus comprises one or more canine immunoglobulin l light chain variable region gene segments. In one aspect, the partly canine immunoglobulin light chain locus comprises one or more canine immunoglobulin k light chain variable region gene segments.

[000128] In one aspect, non-canine mammalian cells are provided that comprise an exogenously introduced, engineered partly canine nucleic acid sequence comprising coding sequences for canine variable regions and non-coding regulatory or scaffold sequences present in the immunoglobulin locus of the mammalian host genome, e.g., mouse genomic non-coding sequences when the host mammal is a mouse. In one aspect, one or more coding sequences for canine variable region gene segments are embedded in non-coding regulatory or scaffold sequences corresponding to those of an immunoglobulin locus in a mammalian host genome. In one aspect, the coding sequences for canine variable region gene segments are embedded in non-coding regulatory or scaffold sequences of a rodent or mouse immunoglobulin locus.

[000129] In one aspect, the partly canine immunoglobulin locus is synthetic and comprises canine VH, D, or JH or VL or JL gene segment coding sequences that are under the control of regulatory elements of the endogenous host. In one aspect, the partly canine immunoglobulin locus comprises canine VH, D, or JH or VL or JL gene segment coding sequences embedded in non-coding regulatory or scaffold sequences corresponding to those of an immunoglobulin locus in a mammalian host genome.

[000130] Methods are also provided for generating a transgenic rodent or rodent ES cell comprising exogenously introduced, engineered partly canine immunoglobulin loci, wherein the resultant transgenic rodent is capable of producing more immunoglobulin comprising l light chain than immunoglobulin comprising k light chain.

[000131] There are many challenges presented when generating a non-canine mammal such as a transgenic mouse or rat, that is capable of producing antigen-specific canine antibodies that are addressed by the constructs and methods described herein, including, but not limited to:

1. How to obtain l:k light chain usage ratio of 90: 10 in an organism such as a mouse or rat that preferentially uses 90% k light chains;

2. Whether mouse B cells can express a large number of dog V, _. gene segments (the dog l locus contains at least 70 functional, unique V, _. gene segments) when the mouse l locus contains only 3 functional V, _. gene segments;

3. How to improve expression and usage of canine V in a non-canine mammal, such as a mouse, in view of the differences in structure between the mouse and dog l light chain loci locus.

a. The mouse l light chain loci locus contains 2 clusters of V gene segment(s), T gene segment(s), and C x exon(s):

i. Ul2-Ul1-Il2-£l2

ii. V i-E. ₃ -Ch. ₃ -E.i-Ch.i; and

b. the dog l locus contains tandem V, _. gene segments upstream

clusters.

4. Whether mouse B cells can develop normally if mouse IgD is expressed with dog VH, in view of the fact that canine IgD is not functional and IgM and IgD are co-expressed as alternatively spliced forms in mouse and rat B cells. Immunoglobulin Loci in mice and dog

[000132] In the humoral immune system, a diverse antibody repertoire is produced by combinatorial and junctional diversity of IGH and IGL chain gene loci by a process termed V(D)J recombination. In the developing B cell, the first recombination event to occur is between one D and one JH gene segment of the heavy chain locus, and the DNA between these two gene segments is deleted. This D-JH recombination is followed by the joining of one VH gene segment from a region upstream of the newly formed DJH complex, forming a rearranged VHDJH exon. All other sequences between the recombined VH and D gene segments of the newly generated VHDJH exon are deleted from the genome of the individual B cell. This rearranged exon is ultimately expressed on the B cell surface as the variable region of the H-chain polypeptide, which is associated with an L-chain polypeptide to form the B cell receptor (BCR).

[000133] The light chain repertoire in the mouse is believed to be shaped by the order of gene rearrangements. The IGK light chain locus on both chromosomes is believed to undergo rearrangements first before the IGL light chain locus on either chromosome becomes receptive for V -J recombination. If an initial k rearrangement is unproductive, additional rounds of secondary rearrangement can proceed, in a process known as receptor editing (Collins and Watson. (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. Immunol. 9:2249.) A process of serial rearrangement of the k chain locus may continue on one chromosome until all possibilities of recombination are exhausted. Recombination will then proceed on the second k chromosome. A failure to produce a productive rearrangement on the second chromosome after multiple rounds of rearrangement will be followed by rearrangement on the l loci (Collins and Watson (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. Immunol. 9:2249.)

[000134] This preferential order of light chain rearrangements is believed to give rise to a light chain repertoire in mouse that is >90% k and <10% l. However, immunoglobulins in the dog immune system are dominated by l light chain usage, which has been estimated to be at least 90% l to <10% k (Arun et al. (1996) Immunohistochemical examination of light-chain expression (l/k ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl Veterinarmed A. 43(9):573-6).

[000135] The murine and canine Ig loci are highly complex in the numbers of features they contain and in how their coding regions are diversified by V(D)J rearrangement; however, this complexity does not extend to the basic details of the structure of each variable region gene segment. The V, D and J gene segments are highly uniform in their compositions and organizations. For example, V gene segments have the following features that are arranged in essentially invariant sequential fashion in immunoglobulin loci: a short transcriptional promoter region (<600bp in length), an exon encoding the 5’ UTR and the majority of the signal peptide for the antibody chain; an intron; an exon encoding a small part of the signal peptide of the antibody chain and the majority of the antibody variable domain, and a 3' recombination signal sequence necessary for V(D)J rearrangement. Similarly, D gene segments have the following necessary and invariant features: a 5' recombination signal sequence, a coding region and a 3' recombination signal sequence. The J gene segments have the following necessary and invariant features: a 5' recombination signal sequence, a coding region and a 3' splice donor sequence.

[000136] The canine genome VH region comprises approximately 39 functional VH, 6 functional D and 5 functional JH gene segments mapping to a 1.46 Mb region of canine chromosome 8. There are also numerous VH pseudogenes and one JH gene segment (IGHJ1) and one D gene segment (IGHD5) that are thought to be non-functional because of non-canonical heptamers in their RSS. (Such gene segments are referred to as Open Reading Frames (ORFs).) Figure 12A provides a schematic diagram of the endogenous canine IGH locus (1201) as well as an expanded view of the IGHC region (1202). The canine immunoglobulin heavy chain variable region locus, which includes VH (1203), D (1204) and JH (1205) gene segments, has all functional genes in the same transcriptional orientation as the constant region genes (1206), with two pseudogenes (IGHV3-4 and IGHV1-4-1) in the reverse transcriptional orientation (not shown). A transcriptional enhancer (1207) and the (1208) m switch region are located within the JH-CP intron. See, Martin et al. (2018) Comprehensive annotation and evolutionary insights into the canine ( Canis lupus familiaris) antigen receptor loci. Immunogenetics. 70:223-236. Among the IGHC genes, Cs (1210) is thought to be non-functional. Moreover, although cDNA clones identified as encoding canine IgGl (1212), IgG2 (1213), IgG3 (1211) and IgG4 (1214) have been isolated (Tang, et al. (2001) Cloning and characterization of cDNAs encoding four different canine immunoglobulin g chains. Vet. Immunol and Immunopath. 80:259 PMID 11457479), only the IgG2 constant region gene has been physically mapped to the canine IGHC locus on chromosome 8. Functional versions of O _m (1209), C _e (1215) and C _a (1216) have also been physically mapped there.

[000137] The sequences of the canine IGHC are in Table 4.

[000138] The canine IGL locus maps to canine chromosome 26, while the canine IGK coding region maps to canine chromosome 17. Figures 12B and 12C provide schematic diagrams of the endogenous canine IGL and IGK loci, respectively.

[000139] The sequences of the canine IGKC and IGLC are in Table 4.

[000140] The canine l locus (1217) is large (2.6 Mbp) with 162 V genes (1218), of which at least 76 are functional. The canine l locus also includes 9 tandem cassettes or J-C units, each containing a L, gene segment and a C exon (1219). See, Martin et al. (2018) Comprehensive annotation and evolutionary insights into the canine ( Canis lupus familiaris) antigen receptor loci. Immunogenetics. 70:223-236.

[000141] The canine k locus (1220) is small (400 Kbp) and has an unusual structure in that eight of the functional VK gene segments are located upstream (1222) and five are located downstream (1226) of the JK (1223) gene segments and CK (1224) exon. The canine upstream VK region has all functional gene segments in the same transcriptional orientation as the JK gene segment and CK exon, with two pseudogenes (IGKV3-3 and IGKV7-2) and one ORF (IGKV4-1) in the reverse transcriptional orientation (not shown). The canine downstream V _K region has all functional gene segments in the opposite transcriptional orientation as the JK gene segment and CK exon and includes six pseudogenes. The Ribose 5-Phosphate Isomerase A (RPIA) gene (1225) is also found in the downstream V _K region, between CK and IGKV2S19. See, Martin et al. (2018) Comprehensive annotation and evolutionary insights into the canine ( Canis lupus familiaris) antigen receptor loci. Immunogenetics. 70:223-236.

[000142] The mouse immunoglobulin k locus is located on chromosome 6. Figure IB provides a schematic diagram of the endogenous mouse IGK locus. The IGK locus (112) spans 3300 Kbp and includes more than 100 variable VK gene segments (113) located upstream of 5 joining (J _K ) gene segments (114) and one constant (C _K ) gene (115). The mouse K locus includes an intronic enhancer (iE _K , 116) located between J _K and C _K that activates k rearrangement and helps maintain the earlier or more efficient rearrangement of K versus l (Inlay et al. (2004) Important Roles for E Protein Binding Sites within the Immunoglobulin k chain intronic enhancer in activating V _K J _K rearrangement. J. Exp. Med. 200(9): 1205-1211). Another enhancer, the 3’ enhancer (3Έ _K , 117) is located 9.1 Kb downstream of the C _K exon and is also involved in k rearrangement and transcription; mutant mice lacking both iE _K and 3Έk have no V _K J _K rearrangements in the k locus (Inlay et al. (2002) Essential roles of the kappa light chain intronic enhancer and 3' enhancer in kappa rearrangement and demethyl ati on. Nature Immunol. 3(5):463-468). However, disrupting the iE _K , for example, by insertion of a neomycin-resistance gene is also sufficient to abolish most V _K J _K rearrangements (Xu et al. (1996) Deletion of the IgK Light Chain Intronic Enhancer/Matrix Attachment Region Impairs but Does Not Abolish V _K J _K Rearrangement).

[000143] The mouse immunoglobulin l locus is located on chromosome 16. Figure 1C provides a schematic diagram of the endogenous mouse IGL locus (118). The organization of the mouse immunoglobulin l locus is different from the mouse immunoglobulin k locus. The locus spans 240 kb, with two clusters comprising 3 functional variable (V ) gene segments (IGLV2, 119; IGLV3, 120 and IGLV1, 123) and 3 tandem cassettes of l joining (J ) gene segments and constant (C.) gene segments (IGLJ2, 121; IGLC2, 122; IGLJ3, 124: IGLC3, 125; IGLJ1, 126; IGLC1, 127) in which the V gene segments are located upstream (5’) from a variable number of J-C tandem cassettes. The locus also contains three transcriptional enhancers (EUA, 128; El, 129; El3-i, 130).

[000144] The partly canine nucleic acid sequence described herein allows the transgenic animal to produce a heavy chain or light chain repertoire comprising canine VH or VL regions, while retaining the regulatory sequences and other elements that can be found within the intervening sequences of the host genome (e.g., rodent) that help to promote efficient antibody production and antigen recognition in the host.

[000145] In one aspect, synthetic, or recombinantly produced, partly canine nucleic acids are engineered to comprise both canine coding sequences and non-canine non-coding regulatory or scaffold sequences of an immunoglobulin VH, VI or V _K locus, or, in some aspects, a combination thereof.

[000146] In one aspect, a transgenic rodent or rodent cell that expresses immunoglobulin with a canine variable region can be generated by inserting one or more canine VH gene segment coding sequences into a VH locus of a rodent heavy chain immunoglobulin locus. In another aspect, a transgenic rodent or rodent cell that expresses immunoglobulin with canine a variable region can be generated by inserting one or more canine VL gene segment coding sequences into a VL locus of a rodent light chain immunoglobulin locus.

[000147] The existence of two light chain loci - k and l - means that a variety of light chain insertion combinations are possible for generating a transgenic rodent or rodent cell that expresses immunoglobulin with canine a variable region, including but not limited to: inserting one or more canine Vi or i, gene segment coding sequences into a rodent Vi locus, inserting one or more canine V _K or J _K gene segment coding sequences into a rodent V _K locus, inserting one or more canine Vi or L gene segment coding sequences into a rodent V _K locus and inserting one or more canine V _K or J _K gene segment coding sequences into a rodent Vi locus.

[000148] The selection and development of a transgenic rodent or rodent cell that expresses partly canine immunoglobulin is complicated by the fact that more than 90% of light chains produced by mice are k and less than 10% are l, whereas more than 90% of light chains produced by dogs are l and less than 10% k and the fact that the canine immunoglobulin l locus is large and includes over 100 Vi gene segments, whereas the mouse immunoglobulin l includes only 3 functional Vi gene segments.

[000149] Since mice produce mainly k LC-containing antibodies, one reasonable method to increase production of l LC-containing partly canine immunoglobulin by the transgenic rodent would be to insert one or more canine Vi or i, gene segment coding sequences into a rodent k locus. However, as shown in the Example 9 below, coupling canine Vi region exon with rodent C _K region exon results in sub-optimal expression of the partly canine immunoglobulin in vitro.

[000150] Provided herein is a transgenic rodent or rodent cell that is capable of expressing immunoglobulin comprising canine variable domains, wherein the transgenic rodent produces more or is more likely to produce immunoglobulin comprising l light chain than immunoglobulin comprising k light chain. While not wishing to be bound by theory, it is believed that a transgenic rodent or rodent cell that produces more, or is more likely to produce, immunoglobulin comprising l light chain will result in a fuller antibody repertoire for the development of therapeutics.

[000151] A transgenic rodent or rodent cell having a genome comprising an engineered partly canine immunoglobulin light chain locus is provided herein. In one aspect, the partly canine immunoglobulin light chain locus comprises canine immunoglobulin l light chain variable region gene segments. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a canine variable domain. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a canine l variable domain. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a canine k variable domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine variable domain and a rodent constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine l variable domain and a rodent l constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine k variable domain and a rodent k constant domain.

[000152] In one aspect, the transgenic rodent or rodent cell produces more, or is more likely to produce, immunoglobulin comprising l light chain than immunoglobulin comprising k light chain. In one aspect, a transgenic rodent is provided in which more l light chain producing cells than k light chain producing cells are likely to be isolated from the rodent. In one aspect, a transgenic rodent is provided that produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% immunoglobulin comprising l light chain. In one aspect, a transgenic rodent cell, or its progeny, is provided that is more likely to produce immunoglobulin with l light chain than immunoglobulin with k light chain. In one aspect, the transgenic rodent cell, or its progeny, has at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and up to about 100%, probability of producing immunoglobulin comprising l light chain. In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent light chain immunoglobulin locus has been deleted and replaced with an engineered partly canine light chain immunoglobulin locus. In one aspect, the transgenic rodent is a mouse.

Immunoglobulin Light Chain Locus

[000153] In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising a recombinantly produced partly canine immunoglobulin variable region locus. In one aspect, the partly canine immunoglobulin variable region locus is a light chain variable region (VL) locus. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more canine V gene segment coding sequences or one or more canine J gene segment coding sequences. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more canine VK gene segment coding sequences or one or more canine JK gene segment coding sequences. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more rodent constant domain genes or coding sequences. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more rodent Cx genes or coding sequences. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more rodent CK genes or coding sequences. In one aspect, an endogenous rodent light chain immunoglobulin locus has been inactivated. In one aspect, an endogenous rodent light chain immunoglobulin locus has been deleted and replaced with an engineered partly canine light chain immunoglobulin locus.

[000154] In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine l variable domain and rodent l constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a canine k variable domain and rodent k constant domain.

[000155] In one aspect, the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising most or all of the V gene segments coding sequences from a canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising at least 20, 30, 40, 50, 60, 70 and up to 76 canine V gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the V gene segment coding sequences from a canine genome.

[000156] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising most or all of the J gene segment coding sequences found in the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 canine J gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the J gene segment coding sequences found in the canine genome.

[000157] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising most or all of the V and J gene segment coding sequences from the canine genome. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the V and J gene segment coding sequences from the canine genome.

[000158] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising most or all of the V _K gene segment coding sequences from the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and up to 14 canine V _K gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the V _K gene segment coding sequences from the canine genome.

[000159] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising most or all of the J _K gene segment coding sequences found in the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising at least 1, 2, 3, 4 or 5 canine J _K gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the J _K gene segment coding sequences found in the canine genome. [000160] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VL locus comprising most or all of the V _K and J _K gene segment coding sequences from the canine genome. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the V _K and J _K gene segment coding sequences from the canine genome.

[000161] In one aspect, the engineered immunoglobulin locus comprises canine VL gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine VL or JL gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the rodent non coding regulatory or scaffold sequences are from a rodent immunoglobulin l light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin k light chain variable region locus. In one aspect, the engineered immunoglobulin locus comprises canine VL and JL gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin l light chain variable region gene locus. In one aspect, the partly canine immunoglobulin locus comprises one or more rodent immunoglobulin l constant region (CL) coding sequences. In one aspect, the partly canine immunoglobulin locus comprises one or more canine VL and JL gene segment coding sequences and one or more rodent immunoglobulin CL coding sequences. In one aspect, the engineered immunoglobulin locus comprises canine VL and JL gene segment coding sequences and one or more rodent CL coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin l light chain variable region gene locus.

[000162] In one aspect, the engineered immunoglobulin locus comprises canine VL or JL gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin k light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine VL or JL gene segment coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine Vx and I _l gene segment coding sequences and one or more rodent immunoglobulin Cx coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin k light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine V and J _/. gene segment coding sequences and one or more rodent immunoglobulin C, _. coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain variable region gene locus.

[000163] In one aspect, one or more canine V gene segment coding sequences are located upstream of one or more Jx gene segment coding sequences, which are located upstream of one or more rodent Cx genes. In one aspect, one or more canine Vx gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more Jx gene segment coding sequences, which are located upstream of one or more rodent lambda Cx genes.

[000164] In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences, one or more canine Jx gene segment coding sequences and one or more rodent Cx genes. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences, one or more canine Jx gene segment coding sequence and one or more rodent Cx region genes, wherein the Vx and Jx gene segment coding sequences and the rodent Cx region genes are inserted into a rodent immunoglobulin k light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences, one or more canine Jx gene segment coding sequence and one or more rodent Cx genes, wherein the Vx and Jx gene segment coding sequences and the rodent (Cx) region genes are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain locus.

[000165] In one aspect, one or more canine Vx gene segment coding sequences are located upstream of one or more Jx gene segment coding sequences, which are located upstream of one or more rodent Cx genes, wherein the Vx and Jxgene segment coding sequences and rodent Cx genes are inserted into a rodent immunoglobulin k light chain locus. In one aspect, one or more canine V, _. gene segment coding sequences are located upstream of one or more Jx gene segment coding sequences, which are located upstream of one or more rodent Cx genes, wherein the V, _. and Jx gene segment coding sequences and rodent CX genes are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain locus.

[000166] In one aspect, the rodent (G coding sequence is selected from a rodent C i, Cx2, or Cx3 coding sequence.

[000167] In one aspect, a transgenic rodent or rodent cell is provided, wherein the engineered immunoglobulin locus comprises a rodent immunoglobulin k locus in which one or more rodent V _K gene segment coding sequences and one or more rodent J _K gene segment coding sequences have been deleted and replaced by one or more canine Vx gene segment coding sequences and one or more Jx gene segment coding sequences, respectively, and in which rodent C _K coding sequences in the locus have been replaced by rodent Cxi, Cx2, or Cx3 coding sequence.

[000168] In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and a rodent CX gene. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent Cx region coding sequence, wherein the Vx gene segment coding sequences and the J-C units are inserted into a rodent immunoglobulin k light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine Vx gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent Cx coding sequence, wherein the Vx gene segment coding sequences and the J-C units are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain locus.

[000169] In one aspect, one or more canine Vx gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more J-C units, wherein each J-C unit comprises a canine Jx gene segment coding sequence and a rodent Cx gene. In one aspect, one or more canine V, _. gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more J-C units, wherein each J-C unit comprises a canine Jx gene segment coding sequence and a rodent Cx coding sequence. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine V, _. gene segment coding sequences located upstream of one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent CX coding sequence, wherein the V, _. gene segment coding sequences and the J-C units are inserted into a rodent immunoglobulin k light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more canine V, _. gene segment coding sequences upstream and in the same transcriptional orientation as one or more J-C units wherein each J-C unit comprises a canine Jx gene segment coding sequence and rodent CX coding sequence, wherein the Vx gene segment coding sequences and the J-C units are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin k light chain locus. In one aspect, the rodent Cx coding sequence is selected from a rodent C i, Cx2, or Cx3 coding sequence.

[000170] In one aspect, the engineered immunoglobulin locus comprises canine VK coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine VK or J _K gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin l light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin k light chain variable region locus. In one aspect, the engineered immunoglobulin locus comprises canine VK and J _K gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin k light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine VK and J _K gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin l light chain variable region gene locus. In one aspect, the partly canine immunoglobulin locus comprises one rodent immunoglobulin CK coding sequences. In one aspect, the partly canine immunoglobulin locus comprises one or more rodent immunoglobulin C,_ coding sequences. In one aspect, the partly canine immunoglobulin locus comprises one or more canine VK and JK gene segment coding sequences and one rodent immunoglobulin CK coding sequences. In one aspect, the engineered immunoglobulin locus comprises canine VK and JK gene segment coding sequences and one rodent immunoglobulin CK coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent k light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises canine VK and JK gene segment coding sequences and one rodent immunoglobulin CK coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin l light chain variable region gene locus.

[000171] While not wishing to be bound by theory, it is believed that inactivating or rendering nonfunctional an endogenous rodent k light chain locus may increase expression of l light chain immunoglobulin from the partly canine immunoglobulin locus. This has been shown to be the case in otherwise conventional mice in which the k light chain locus has been inactivated in the germline (Zon, et al. (1995) Subtle differences in antibody responses and hypermutation of l light chains in mice with a disrupted k constant region. Eur. J. Immunol. 25:2154-2162). In one aspect, inactivating or rendering nonfunctional an endogenous rodent k light chain locus may increase the relative amount of immunoglobulin comprising l light chain relative to the amount of immunoglobulin comprising k light chain produced by the transgenic rodent or rodent cell.

[000172] In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin k light chain locus is deleted, inactivated, or made nonfunctional. In one aspect, the endogenous rodent immunoglobulin k light chain locus is inactivated or made nonfunctional by one or more of the following deleting or mutating all endogenous rodent VK gene segment coding sequences; deleting or mutating all endogenous rodent J _K gene segment coding sequences; deleting or mutating the endogenous rodent CK coding sequence; deleting, mutating, or disrupting the endogenous intronic k enhancer (iE _K ) and 3’ enhancer sequence (3Έ _K ); or a combination thereof. [000173] In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin l light chain variable domain is deleted, inactivated, or made nonfunctional. In one aspect, the endogenous rodent immunoglobulin l light chain variable domain is inactivated or made nonfunctional by one or more of the following: deleting or mutating all endogenous rodent V _K gene segments; deleting or mutating all endogenous rodent I _l gene segments; deleting or mutating all endogenous rodent C,_ coding sequences; or a combination thereof.

[000174] In one aspect, the partly canine immunoglobulin locus comprises rodent regulatory or scaffold sequences, including, but not limited to enhancers, promoters, splice sites, introns, recombination signal sequences, and combinations thereof. In one aspect, the partly canine immunoglobulin locus comprises rodent l regulatory or scaffold sequences. In one aspec, the partly canine immunoglobulin locus comprises rodent k regulatory or scaffold sequences.

[000175] In one aspect, the partly canine immunoglobulin locus includes a promoter to drive gene expression. In one aspect, the partly canine immunoglobulin locus includes a k V- region promoter. In one aspect, the partly canine immunoglobulin locus includes a l V- region promoter. In one aspect, the partly canine immunoglobulin locus includes a l V- region promoter to drive expression of one or more l LC gene coding sequences created after V, _. to I _l gene segment rearrangement. In one aspect, the partly canine immunoglobulin locus includes a l V-region promoter to drive expression of one or more K LC gene coding sequences created after V _K to JK gene segment rearrangement. In one aspect, the partly canine immunoglobulin locus includes a k V-region promoter to drive expression of one or more l LC gene coding sequences created after V, _. to L. gene segment rearrangement. In one aspect, the partly canine immunoglobulin locus includes a k V- region promoter to drive expression of one or more k LC gene coding sequences created after V _K to JK gene segment rearrangement.

[000176] In one aspect, the partly canine immunoglobulin locus includes one or more enhancers. In one aspect, the partly canine immunoglobulin locus includes a mouse k ΐEk or 3Έk enhancer. In one aspect, the partly canine immunoglobulin locus includes one or more U _l or I _l gene segment coding sequences and a moue k iE _K or 3Έ _K enhancer. In one aspect, the partly canine immunoglobulin locus includes one or more V _K or J _K gene segment coding sequences and a k ΐEk or 3Έk enhancer.

Immunoglobulin Heavy Chain Locus

[000177] In one aspect, a transgenic rodent or rodent cell has a genome comprising a recombinantly produced partly canine immunoglobulin heavy chain variable region (VH) locus. In one aspect, the partly canine immunoglobulin variable region locus comprises one or more canine VH, D or JH gene segment coding sequences. In one aspect, the partly canine immunoglobulin heavy chain variable region locus comprises one or more rodent constant domain (CH) genes or coding sequences. In one aspect, an endogenous rodent heavy chain immunoglobulin locus has been inactivated. In one aspect, an endogenous rodent heavy chain immunoglobulin locus has been deleted and replaced with an engineered partly canine heavy chain immunoglobulin locus.

[000178] In one aspect, the synthetic H chain DNA segment contains the ADAM6A or ADAM6B gene needed for male fertility, Pax-5 -Activated Intergenic Repeats (PAIR) elements involved in Igh locus contraction and CTCF binding sites from the heavy chain intergenic control region 1, involved in regulating normal VDJ rearrangement ((Proudhon, et al., Adv. Immunol., 128: 123-182 (2015)), or various combinations thereof. The locations of these endogenous non-coding regulatory and scaffold sequences in the mouse IGH locus are depicted in FIG 1, which illustrates from left to right: the -100 functional heavy chain variable region gene segments (101); PAIR, Pax-5 Activated Intergenic Repeats involved in IGH locus contraction for VDJ recombination (102); ADAM6A or ADAM6B, a disintegrin and metallopeptidase domain 6A gene required for male fertility (103); Pre-D region, a 21609 bp fragment upstream of the most distal DH gene segment, IGHD-5 D (104); Intergenic Control Region 1 (IGCR1) that contains CTCF insulator sites to regulate VH gene segment usage (106); D, diversity gene segments (10-15 depending on the mouse strain) (105); four joining JH gene segments (107); E _m , the intronic enhancer involved in VDJ recombination (108); Sn, the m switch region for isotype switching (109); eight heavy chain constant region genes: Cn, Ca, CV, C _7i , CVb, C2 _Ya/c , C _e , and Ca (110); 3' Regulatory Region (3'RR) that controls isotype switching and somatic hypermutation (111). FIG. 1 A is modified from a figure taken from Proudhon, et al., Adv. Immunol., 128: 123-182 (2015). [000179] In one aspect, the engineered partly canine region to be integrated into a mammalian host cell comprises all or a substantial number of the known canine VH gene segments. In some instances, however, it may be desirable to use a subset of such VH gene segments, and in specific instances even as few as one canine VH coding sequence may be introduced into the cell or the animal.

[000180] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH gene segment coding sequences from the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising at least 20, 30 and up to 39 functional canine VH gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH gene segment coding sequences from the canine genome.

[000181] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH gene segment coding sequences from the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising at least 20, 30, 40, 50, 60, 70 and up to 80 canine VH gene segment coding sequences. In this aspect the VH gene segment pseudogenes are reverted to restore their functionality, e.g., by mutating an in-frame stop codon into a functional codon, using methods well known in the art. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH gene segment coding sequences from the canine genome.

[000182] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising most or all of the D gene segment coding sequences found in the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, 3, 4, 5 and up to 6 canine D gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the D gene segment coding sequences found in the canine genome. [000183] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising most or all of the JH gene segment coding sequences found in the canine genome. In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, 3, 4, 5 and up to 6 canine JH gene segment coding sequences. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 75%, and up to 100% of JH gene segment coding sequences found in the canine genome.

[000184] In one aspect, the engineered partly canine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH, D and JH gene segment coding sequences from the canine genome. In one aspect the engineered partly canine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH, D and JH gene segment coding sequences from the canine genome.

[000185] In one aspect, a transgenic rodent or rodent cell is provided that includes an engineered partly canine immunoglobulin heavy chain locus comprising canine immunoglobulin heavy chain variable region gene coding sequences and non-coding regulatory or scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the engineered canine immunoglobulin heavy chain locus comprises canine VH, D or JH gene segment coding sequences. In one aspect, the engineered canine immunoglobulin heavy chain locus comprises canine VH, D or JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain locus.

[000186] In one aspect, non-canine mammals and mammalian cells comprising an engineered partly canine immunoglobulin locus that comprises coding sequences of canine VH, canine D, and canine JH genes are provided that further comprises non-coding regulatory and scaffold sequences, including pre-D sequences, based on the endogenous IGH locus of the non-canine mammalian host. In certain aspects, the exogenously introduced, engineered partly canine region can comprise a fully recombined V(D)J exon.

[000187] In one aspect, the transgenic non-canine mammal is a rodent, for example, a mouse, comprising an exogenously introduced, engineered partly canine immunoglobulin locus comprising codons for multiple canine VH, canine D, and canine JH genes with intervening sequences, including a pre-D region, based on the intervening (non-coding regulatory or scaffold) sequences in the rodent. In one aspect, the transgenic rodent further comprises partly canine IGL loci comprising coding sequences of canine V _K or V _L genes and J _K or I _l genes, respectively, in conjunction with their intervening (non-coding regulatory or scaffold) sequences corresponding to the immunoglobulin intervening sequences present in the IGL loci of the rodent.

[000188] In an exemplary embodiment, as set forth in more detail in the Examples section, the entire endogenous VH immunoglobulin locus of the mouse genome is deleted and subsequently replaced with a partly canine immunoglobulin locus comprising 39 canine VH gene segments containing interspersed non-coding sequences corresponding to the non coding sequences of the J558 VH locus of the mouse genome. The complete, exogenously introduced, engineered immunoglobulin locus further comprises canine D and JH gene segments, as well as the mouse pre-D region. Thus, the canine VH, D and JH codon sequences are embedded in the rodent intergenic and intronic sequences.

Preparation of a Partly Canine Immunoglobulin Locus

[000189] In one aspect, an endogenous immunoglobulin locus variable region of a non canine mammal, such as a rodent, for example a rat or mouse, which contains VH, D and JH or VL and JL gene segments, is deleted using site-specific recombinases and replaced with an engineered partly canine immunoglobulin locus. In one aspect, the partly canine immunoglobulin locus is inserted into the genome of the host animal as a single nucleic acid or cassette. Because a cassette that includes the partly canine immunoglobulin locus is used to replace the endogenous immunoglobulin locus variable region, the canine coding sequences can be inserted into the host genome in a single insertion step, thus providing a rapid and straightforward process for obtaining a transgenic animal.

[000190] In one aspect, the engineered partly canine immunoglobulin locus variable region is prepared by deleting murine VH, D and JH or VL and JL coding sequences from a mouse immunoglobulin locus variable region and replacing the murine coding sequences with canine coding sequences. In one aspect, the non-coding flanking sequences of the murine immunoglobulin locus, which include regulatory sequences and other elements, are left intact. [000191] In one aspect, the nucleotide sequence for the engineered partly canine immunoglobulin locus is prepared in silico and the locus is synthesized using known techniques for gene synthesis. In one aspect, coding sequences from a canine immunoglobulin variable region locus and sequences of the host animal immunoglobulin locus are identified using a search tool such as BLAST (Basic Local Alignment Search Tool). After obtaining the genomic sequences of the host immunoglobulin locus and the coding sequences of the canine immunoglobulin variable region locus, the host coding sequences can be replaced in silico with the canine coding sequences using known computational approaches to locate and delete the endogenous host animal immunoglobulin coding segments and replace the coding sequences with canine coding sequences, leaving the endogenous regulatory and flanking sequences intact.

Homologous Recombination

[000192] In one aspect, a combination of homologous recombination and site-specific recombination is used to create the cells and animals described herein. In some embodiments, a homology targeting vector is first used to introduce the sequence-specific recombination sites into the mammalian host cell genome at a desired location in the endogenous immunoglobulin loci. In one aspect, in the absence of a recombinase protein, the sequence-specific recombination site inserted into the genome of a mammalian host cell by homologous recombination does not affect expression and amino acid codons of any genes in the mammalian host cell. This approach maintains the proper transcription and translation of the immunoglobulin genes which produce the desired antibody after insertion of recombination sites and, optionally, any additional sequence such as a selectable marker gene. However, in some cases it is possible to insert a recombinase site and other sequences into an immunoglobulin locus sequence such that an amino acid sequence of the antibody molecule is altered by the insertion, but the antibody still retains sufficient functionality for the desired purpose. Examples of such codon-altering homologous recombination may include the introduction of polymorphisms into the endogenous locus and changing the constant region exons so that a different isotype is expressed from the endogenous locus. In one aspect, the immunoglobulin locus includes one or more of such insertions. [000193] In one aspect, the homology targeting vector can be utilized to replace certain sequences within the endogenous genome as well as to insert certain sequence-specific recombination sites and one or more selectable marker genes into the host cell genome. It is understood by those of ordinary skill in the art that a selectable marker gene as used herein can be exploited to weed out individual cells that have not undergone homologous recombination and cells that harbor random integration of the targeting vector.

[000194] Exemplary methodologies for homologous recombination are described in U. S. Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of which is incorporated by reference in its entirety.

Site/Sequence-Specific Recombination

[000195] Site/sequence-specific recombination differs from general homologous recombination in that short specific DNA sequences, which are required for recognition by a recombinase, are the only sites at which recombination occurs. Depending on the orientations of these sites on a particular DNA strand or chromosome, the specialized recombinases that recognize these specific sequences can catalyze i) DNA excision or ii) DNA inversion or rotation. Site-specific recombination can also occur between two DNA strands if these sites are not present on the same chromosome. A number of bacteriophage- and yeast-derived site-specific recombination systems, each comprising a recombinase and specific cognate sites, have been shown to work in eukaryotic cells and are therefore applicable for use in connection with the methods described herein, and these include the bacteriophage PI Cre/lox, yeast FLP-FRT system, and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are described, e.g., in U.S. Pat. Nos. 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317, each of which is incorporated herein by reference to teach methods of using such recombinases.

[000196] Other systems of the tyrosine family of site-specific recombinases such as bacteriophage lambda integrase, HK2022 integrase, and in addition systems belonging to the separate serine family of recombinases such as bacteriophage phiC31, R4Tp901 integrases are known to work in mammalian cells using their respective recombination sites, and are also applicable for use in the methods described herein. [000197] Since site-specific recombination can occur between two different DNA strands, site-specific recombination occurrence can be utilized as a mechanism to introduce an exogenous locus into a host cell genome by a process called recombinase-mediated cassette exchange (RMCE). The RMCE process can be exploited by the combined usage of wild- type and mutant sequence-specific recombination sites for the same recombinase protein together with negative selection. For example, a chromosomal locus to be targeted may be flanked by a wild-type LoxP site on one end and by a mutant LoxP site on the other. Likewise, an exogenous vector containing a sequence to be inserted into the host cell genome may be similarly flanked by a wild-type LoxP site on one end and by a mutant LoxP site on the other. When this exogenous vector is transfected into the host cell in the presence of Cre recombinase, Cre recombinase will catalyze RMCE between the two DNA strands, rather than the excision reaction on the same DNA strands, because the wild-type LoxP and mutant LoxP sites on each DNA strand are incompatible for recombination with each other. Thus, the LoxP site on one DNA strand will recombine with a LoxP site on the other DNA strand; similarly, the mutated LoxP site on one DNA strand will only recombine with a likewise mutated LoxP site on the other DNA strand.

[000198] In one aspect, combined variants of the sequence-specific recombination sites are used that are recognized by the same recombinase for RMCE. Examples of such sequence- specific recombination site variants include those that contain a combination of inverted repeats or those which comprise recombination sites having mutant spacer sequences. For example, two classes of variant recombinase sites are available to engineer stable Cre-loxP integrative recombination. Both exploit sequence mutations in the Cre recognition sequence, either within the 8 bp spacer region or the 13 -bp inverted repeats. Spacer mutants such as lox511 (Hoess, et ah, Nucleic Acids Res, 14:2287-2300 (1986)), lox5171 and lox2272 (Lee and Saito, Gene, 216:55-65 (1998)), m2, m3, m7, and mi l (Langer, et ah, Nucleic Acids Res, 30:3067-3077 (2002)) recombine readily with themselves but have a markedly reduced rate of recombination with the wild-type site. This class of mutants has been exploited for DNA insertion by RMCE using non-interacting Cre-Lox recombination sites and non-interacting FLP recombination sites (Baer and Bode, Curr Opin Biotechnol, 12:473-480 (2001); Albert, et ah, Plant J, 7:649-659 (1995); Seibler and Bode, Biochemistry, 36:1740-1747 (1997); Schlake and Bode, Biochemistry, 33: 12746-12751 (1994)).

[000199] Inverted repeat mutants represent the second class of variant recombinase sites. For example, LoxP sites can contain altered bases in the left inverted repeat (LE mutant) or the right inverted repeat (RE mutant). An LE mutant, lox71, has 5 bp on the 5' end of the left inverted repeat that is changed from the wild type sequence to TACCG (Araki, et al, Nucleic Acids Res, 25:868-872 (1997)). Similarly, the RE mutant, lox66, has the five 3'- most bases changed to CGGTA. Inverted repeat mutants are used for integrating plasmid inserts into chromosomal DNA with the LE mutant designated as the "target" chromosomal loxP site into which the "donor" RE mutant recombines. Post-recombination, loxP sites are located in cis, flanking the inserted segment. The mechanism of recombination is such that post-recombination one loxP site is a double mutant (containing both the LE and RE inverted repeat mutations) and the other is wild type (Lee and Sadowski, Prog Nucleic Acid Res Mol Biol, 80: 1-42 (2005); Lee and Sadowski, J Mol Biol, 326:397-412 (2003)). The double mutant is sufficiently different from the wild-type site that it is unrecognized by Cre recombinase and the inserted segment is not excised.

[000200] In certain aspects, sequence-specific recombination sites can be introduced into introns, as opposed to coding nucleic acid regions or regulatory sequences. This avoids inadvertently disrupting any regulatory sequences or coding regions necessary for proper antibody expression upon insertion of sequence-specific recombination sites into the genome of the animal cell.

[000201] Introduction of the sequence-specific recombination sites may be achieved by conventional homologous recombination techniques. Such techniques are described in references such as e.g., Sambrook and Russell (2001) (Molecular cloning: a laboratory manual 3rd ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) and Nagy, A. (2003). (Manipulating the mouse embryo: a laboratory manual, 3rd ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Renault and Duchateau, Eds. (2013) (Site-directed insertion of transgenes. Topics in Current Genetics 23. Springer). Tsubouchi, H. Ed. (2011) (DNA recombination, Methods and Protocols. Humana Press).

[000202] Specific recombination into the genome can be facilitated using vectors designed for positive or negative selection as known in the art. In order to facilitate identification of cells that have undergone the replacement reaction, an appropriate genetic marker system may be employed and cells selected by, for example, use of a selection tissue culture medium. However, in order to ensure that the genome sequence is substantially free of extraneous nucleic acid sequences at or adjacent to the two end points of the replacement interval, desirably the marker system/gene can be removed following selection of the cells containing the replaced nucleic acid.

[000203] In one aspect, cells in which the replacement of all or part of the endogenous immunoglobulin locus has taken place are negatively selected against upon exposure to a toxin or drug. For example, cells that retain expression of HSV-TK can be selected against by using nucleoside analogues such as ganciclovir. In another aspect, cells comprising the deletion of the endogenous immunoglobulin locus may be positively selected for by use of a marker gene, which can optionally be removed from the cells following or as a result of the recombination event. A positive selection system that may be used is based on the use of two non-functional portions of a marker gene, such as HPRT, that are brought together through the recombination event. These two portions are brought into functional association upon a successful replacement reaction being carried out and wherein the functionally reconstituted marker gene is flanked on either side by further sequence- specific recombination sites (which are different from the sequence-specific recombination sites used for the replacement reaction), such that the marker gene can be excised from the genome, using an appropriate site-specific recombinase.

[000204] The recombinase may be provided as a purified protein, or as a protein expressed from a vector construct transiently transfected into the host cell or stably integrated into the host cell genome. Alternatively, the cell may be used first to generate a transgenic animal, which then may be crossed with an animal that expresses said recombinase.

[000205] Because the methods described herein can take advantage of two or more sets of sequence-specific recombination sites within the engineered genome, multiple rounds of RMCE can be exploited to insert the partly canine immunoglobulin variable region genes into a non-canine mammalian host cell genome.

[000206] Although not yet routine for the insertion of large DNA segments, CRISPR-Cas technology is another method to introduce the chimeric canine Ig locus. Generation of Transgenic Animals

[000207] In one aspect, methods for the creation of transgenic animals, for example rodents, such as mice, are provided that comprise the introduced partly canine immunoglobulin locus.

[000208] In one aspect, the host cell utilized for replacement of the endogenous immunoglobulin genes is an embryonic stem (ES) cell, which can then be utilized to create a transgenic mammal. In one aspect, the host cell is a cell of an early stage embryo. In one aspect, the host cell is a pronuclear stage embryo or zygote. Thus, in accordance with one aspect, the methods described herein further comprise: isolating an embryonic stem cell or a cell of an early stage embryo such as a pronuclear stage embryo or zygote, which comprises the introduced partly canine immunoglobulin locus and using said ES cell to generate a transgenic animal that contains the replaced partly canine immunoglobulin locus.

Methods of Use

[000209] In one aspect, a method of producing antibodies comprising canine variable regions is provided. In one aspect, the method includes providing a transgenic rodent or rodent cell described herein and isolating antibodies comprising canine variable regions expressed by the transgenic rodent. In one aspect, a method of producing monoclonal antibodies comprising canine variable regions is provided. In one aspect, the method includes providing B-cells from a transgenic rodent or cell described herein, immortalizing the B-cells; and isolating antibodies comprising canine variable domains expressed by the immortalized B-cells.

[000210] In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine HC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse HC constant domains. These can be of any isotype, IgM, IgD, IgGl, IgG2a/c, IgG2b, IgG3, IgE or IgA.

[000211] In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine HC variable domains and mouse HC constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine LC variable domains and mouse LC constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine HC variable domains and canine LC variable domains and mouse HC constant domains and mouse LC constant domains.

[000212] In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine l LC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse l constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine l LC variable domains and mouse l constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine k LC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse k constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise canine k LC variable domains and mouse k constant domains.

[000213] In one aspect, a method of producing antibodies or antigen binding fragments comprising canine variable regions is provided. In one aspect, the method includes providing a transgenic rodent or cell described herein and isolating antibodies comprising canine variable regions expressed by the transgenic rodent or rodent cell. In one aspect, the variable regions of the antibody expressed by the transgenic rodent or rodent cell are sequenced. Antibodies comprising canine variable regions obtained from the antibodies expressed by the transgenic rodent or rodent cell can be recombinantly produced using known methods.

[000214] In one aspect, a method of producing an immunoglobulin specific to an antigen of interest is provided. In one aspect, the method includes immunizing a transgenic rodent as described herein with the antigen and isolating immunoglobulin specific to the antigen expressed by the transgenic rodent or rodent cell. In one aspect, the variable domains of the antibody expressed by the rodent or rodent cell are sequenced and antibodies comprising canine variable regions that specifically bind the antigen of interest are recombinantly produced using known methods. In one aspect, the recombinantly produced antibody or antigen binding fragment comprises canine HC and LC, k or l, constant domains. Incorporation by Reference

[000215] All references cited herein, including patents, patent applications, papers, text books and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety for all purposes.

EXAMPLES

[000216] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[000217] Efforts have been made to ensure accuracy with respect to terms and numbers used (e.g., vectors, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

[000218] The examples illustrate targeting by both a 5' vector and a 3' vector that flank a site of recombination and introduction of synthetic DNA. It will be apparent to one skilled in the art upon reading the specification that the 5' vector targeting can take place first followed by the 3', or the 3' vector targeting can take place first followed by the 5' vector. In some circumstances, targeting can be carried out simultaneously with dual detection mechanisms. Example 1: Introduction of an Engineered Partly Canine Immunoglobulin Variable Region Gene Locus into the Immunoglobulin H Chain Variable Region Gene Locus of a Non-Canine Mammalian Host Cell Genome

[000219] An exemplary method illustrating the introduction of an engineered partly canine immunoglobulin locus into the genomic locus of a non-mammalian ES cell is illustrated in more detail in FIGS. 2-6. In FIG. 2, a homology targeting vector (201) is provided comprising a puromycin phosphotransferase-thymidine kinase fusion protein (puro-TK) (203) flanked by two different recombinase recognition sites (e.g., FRT (207) and loxP (205) for Flp and Cre, respectively) and two different mutant sites (e.g., modified mutant FRT (209) and mutant loxP (211)) that lack the ability to recombine with their respective wild-type counterparts/sites (i.e., wild-type FRT (207) and wild-type loxP (205)). The targeting vector comprises a diphtheria toxin receptor (DTR) cDNA (217) for use in negative selection of cells containing the introduced construct in future steps. The targeting vector also optionally comprises a visual marker such as a green fluorescent protein (GFP) (not shown). The regions 213 and 215 are homologous to the 5' and 3' portions, respectively, of a contiguous region (229) in the endogenous non-canine locus that is 5' of the genomic region comprising the endogenous non-canine VH gene segments (219). The homology targeting vector (201) is introduced (202) into the ES cell, which has an immunoglobulin locus (231) comprising endogenous VH gene segments (219), the pre-D region (221), the D gene segments (223), JH gene segments (225), and the immunoglobulin constant gene region genes (227). The site-specific recombination sequences and the DTR cDNA from the homology targeting vector (201) are integrated (204) into the non-canine genome at a site 5' of the endogenous mouse VH gene locus, resulting in the genomic structure illustrated at 233. The ES cells that do not have the exogenous vector (201) integrated into their genome can be selected against (killed) by including puromycin in the culture medium; only the ES cells that have stably integrated the exogenous vector (201) into their genome and constitutively express the puro-TK gene are resistant to puromycin.

[000220] FIG. 3 illustrates effectively the same approach as FIG. 2, except that an additional set of sequence-specific recombination sites is added, e.g., a Rox site (331) and a modified Rox site (335) for use with the Dre recombinase. In FIG. 3, a homology targeting vector (301) is provided comprising a puro-TK fusion protein (303) flanked by wild type recombinase recognition sites for FRT (307), loxP (305), and Rox (331) and mutant sites for FRT (309) loxP (311) and Rox (335) recombinases that lack the ability to recombine with the wild-type sites 307, 305 and 331, respectively. The targeting vector also comprises a diphtheria toxin receptor (DTR) cDNA (317). The regions 313 and 315 are homologous to the 5' and 3' portions, respectively, of a contiguous region (329) in the endogenous non-canine locus that is 5' of the genomic region comprising the endogenous mouse VH gene segments (319). The homology targeting is introduced (302) into the mouse immunoglobulin locus (339), which comprises the endogenous VH gene segments (319), the pre-D region (321), the D gene segments (323), JH (325) gene segments, and the constant region genes (327) of the Igh locus. The site-specific recombination sequences and the DTR cDNA (317) in the homology targeting vector (301) are integrated (304) into the mouse genome at a site 5' of the endogenous mouse VH gene locus, resulting in the genomic structure illustrated at 333.

[000221] As illustrated in FIG. 4, a second homology targeting vector (401) is provided comprising an optional hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (435) that can be used for positive selection in HPRT-deficient ES cells; a neomycin resistance gene (437); recombinase recognition sites FRT (407) and loxP (405), for Flp and Cre, respectively, which have the ability to recombine with FRT (407) and loxP (405) sites previously integrated into the mouse genome from the first homology targeting vector. The previous homology targeting vector also includes mutant FRT site (409), mutant loxP site (411), a puro-TK fusion protein (403), and a DTR cDNA at a site 5' of the endogenous mouse VH gene locus (419). The regions 429 and 439 are homologous to the 5' and 3' portions, respectively, of a contiguous region (441) in the endogenous mouse non-canine locus that is downstream of the endogenous JH gene segments (425) and upstream of the constant region genes (427). The homology targeting vector is introduced (402) into the modified mouse immunoglobulin locus (431), which comprises the endogenous VH gene segments (419), the pre-D region (421), the D gene segments (423) the JH gene segments (425), and the constant region genes (427). The site-specific recombination sequences (407, 405), the HPRT gene (435) and a neomycin resistance gene (437) of the homology targeting vector are integrated (404) into the mouse genome upstream of the endogenous mouse constant region genes (427), resulting in the genomic structure illustrated at 433. [000222] Once the recombination sites are integrated into the mammalian host cell genome, the endogenous region of the immunoglobulin domain is then subjected to recombination by introducing one of the recombinases corresponding to the sequence-specific recombination sites integrated into the genome, e.g., either Flp or Cre. Illustrated in FIG. 5 is a modified Igh locus of the mammalian host cell genome comprising two integrated DNA fragments. One fragment comprising mutant FRT site (509), mutant LoxP site (511), puro-TK gene (503), wild-type FRT site (507), and wild-type LoxP site (505), and DTR cDNA (517) is integrated upstream of the VH gene locus (519). The other DNA fragment comprising HPRT gene (535), neomycin resistance gene (537), wild-type FRT site (507), and wild-type LoxP site (505) is integrated downstream of the pre-D (521), D (523) and JH (525) gene loci, but upstream of the constant region genes (527). In the presence of Flp or Cre (502), all the intervening sequences between the wild-type FRT or wild-type LoxP sites including the DTR gene (517), the endogenous IGH variable region gene loci (519, 521, 525), and the HPRT (535) and neomycin resistance (537) genes are deleted, resulting in a genomic structure illustrated at 539. The procedure depends on the second targeting having occurred on the same chromosome rather than on its homolog (i.e., in c/s rather than in irons ^' ). If the targeting occurs in cis as intended, the cells are not sensitive to negative selection after Cre- or Flp-mediated recombination by diphtheria toxin introduced into the media, because the DTR gene which causes sensitivity to diphtheria toxin in rodents should be absent (deleted) from the host cell genome. Likewise, ES cells that harbor random integration of the first or second targeting vector(s) are rendered sensitive to diphtheria toxin by presence of the undeleted DTR gene.

[000223] ES cells that are insensitive to diphtheria toxin are then screened for the deletion of the endogenous variable region gene loci. The primary screening method for the deleted endogenous immunoglobulin locus can be carried out by Southern blotting, or by polymerase chain reaction (PCR) followed by confirmation with a secondary screening technique such as Southern blotting.

[000224] FIG. 6 illustrates introduction of the engineered partly canine sequence into a non canine genome previously modified to delete part of the endogenous Igh locus (VH, D and JH) that encodes the heavy chain variable region domains as well as all the intervening sequences between the VH and JH gene locus. A site-specific targeting vector (629) comprising partly canine VH gene locus (619), endogenous non-canine pre-D gene region (621), partly canine D gene locus (623), partly canine JH gene locus (625), as well as flanking mutant FRT (609), mutant LoxP (611), wild-type FRT (607), and wild-type LoxP (605) sites is introduced (602) into the host cell. Specifically, the partly canine VH locus (619) comprises 39 functional canine VH coding sequences in conjunction with the intervening sequences based on the endogenous non-canine genome sequences; the pre-D region (621) comprises a 21.6 kb mouse sequence with significant homology to the corresponding region of the endogenous canine IGH locus; the D gene locus (623) comprises codons of 6 D gene segments embedded in the intervening sequences surrounding the endogenous non-canine D gene segments; and the JH gene locus (625) comprises codons of 6 canine JH gene segments embedded in the intervening sequences based on the endogenous non-canine genome. The IGH locus (601) of the host cell genome has been previously modified to delete all the VH, D, and JH gene segments including the intervening sequences as described in FIG. 5. As a consequence of this modification, the endogenous non-canine host cell Igh locus (601) is left with a puro-TK fusion gene (603), which is flanked by a mutant FRT site (609) and a mutant LoxP site (611) upstream as well as a wild-type FRT (607) and a wild-type LoxP (605) downstream. Upon introduction of the appropriate recombinase (604), the partly canine immunoglobulin locus is integrated into the genome upstream of the endogenous non-canine constant region genes (627), resulting in the genomic structure illustrated at 631.

[000225] The sequences of the canine VH, D and JH gene segment coding regions are in Table 1

[000226] Primary screening procedure for the introduction of the partly canine immunoglobulin locus can be carried out by Southern blotting, or by PCR followed by confirmation with a secondary screening method such as Southern blotting. The screening methods are designed to detect the presence of the inserted VH, D and JH gene loci, as well as all the intervening sequences. Example 2: Introduction of an Engineered Partly Canine Immunoglobulin Variable Region Gene Locus Comprising Additional Non-Coding Regulatory or Scaffold

Sequences into the Immunoglobulin H Chain Variable Region Gene Locus of a Non- Canine Mammalian Host Cell Genome

[000227] In certain aspects, the partly canine immunoglobulin locus comprises the elements as described in Example 1, but with additional non-coding regulatory or scaffold sequences e.g., sequences strategically added to introduce additional regulatory sequences, to ensure the desired spacing within the introduced immunoglobulin locus, to ensure that certain coding sequences are in adequate juxtaposition with other sequences adjacent to the replaced immunoglobulin locus, and the like. FIG. 7 illustrates the introduction of a second exemplary engineered partly canine sequence into the modified non-canine genome as produced in FIGS. 2-5 and described in Example 1 above.

[000228] FIG. 7 illustrates introduction of the engineered partly canine sequence into the mouse genome previously modified to delete part of the endogenous non-canine IGH locus (VH, D and JH) that encodes the heavy chain variable region domains as well as all the intervening sequences between the endogenous VH and JH gene loci. A site-specific targeting vector (731) comprising an engineered partly canine immunoglobulin locus to be inserted into the non-canine host genome is introduced (702) into the genomic region (701). The site-specific targeting vector (731) comprising a partly canine VH gene locus (719), mouse pre-D region (721), partly canine D gene locus (723), partly canine JH gene locus (725), PAIR elements (741), as well as flanking mutant FRT (709), mutant LoxP (711) wild-type FRT (707) and wild-type LoxP (705) sites is introduced (702) into the host cell. Specifically, the engineered partly canine VH gene locus (719) comprises 80 canine VH gene segment coding regions in conjunction with intervening sequences based on the endogenous non- canine genome sequences; the pre-D region (721) comprises a 21.6 kb non- canine sequence present upstream of the endogenous non-canine genome; the D region (723) comprises codons of 6 canine D gene segments embedded in the intervening sequences surrounding the endogenous non-canine D gene segments; and the JH gene locus (725) comprises codons of 6 canine JH gene segments embedded in the intervening sequences based on the endogenous non- canine genome sequences. The IGH locus (701) of the host cell genome has been previously modified to delete all the VH, D and JH gene segments including the intervening sequences as described in relation to FIG. 5. As a consequence of this modification, the endogenous non- canine Igh locus (701) is left with a puro-TK fusion gene (703), which is flanked by a mutant FRT site (709) and a mutant LoxP site (711) upstream as well as a wild-type FRT (707) and a wild-type LoxP (705) downstream. Upon introduction of the appropriate recombinase (704), the engineered partly canine immunoglobulin locus is integrated into the genome upstream of the endogenous mouse constant region genes (727), resulting in the genomic structure illustrated at 729.

[000229] The primary screening procedure for the introduction of the engineered partly canine immunoglobulin region can be carried out by Southern blotting, or by PCR with confirmation by a secondary screening method such as Southern blotting. The screening methods are designed to detect the presence of the inserted PAIR elements, the VH, D and JH gene loci, as well as all the intervening sequences.

Example 3: Introduction of an Engineered Partly Canine Immunoglobulin Locus into the Immunoglobulin Heavy Chain Gene Locus of a Mouse Genome

[000230] A method for replacing a portion of a mouse genome with an engineered partly canine immunoglobulin locus is illustrated in FIG. 8. This method uses introduction of a first site-specific recombinase recognition sequence into the mouse genome followed by the introduction of a second site-specific recombinase recognition sequence into the mouse genome. The two sites flank the entire clusters of endogenous mouse VH, D and JH region gene segments. The flanked region is deleted using the relevant site-specific recombinase, as described herein.

[000231] The targeting vectors (803, 805) employed for introducing the site-specific recombinase sequences on either side of the VH (815), D (817) and JH (819) gene segment clusters and upstream of the constant region genes (821) in the wild-type mouse immunoglobulin locus (801) include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This mutant modified site (e.g., lox5171) is positioned in the targeting vector such that after deletion of the endogenous VH, DH and JH gene segments (802) it can be used for a second site-specific recombination event in which a non-native piece of DNA is moved into the modified IGH locus by RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising both canine and non canine sequences (809).

[000232] Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (803) comprises mouse genomic DNA taken from the 5' end of the Igh locus, upstream of the most distal VH gene segment. The other vector (805) comprises mouse genomic DNA taken from within the locus downstream of the JH gene segments.

[000233] The key features of the 5' vector (803) in order from 5' to 3' are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (823); 4.5 Kb of mouse genomic DNA mapping upstream of the most distal VH gene segment in the Igh locus (825); a FRT recognition sequence for the Flp recombinase (827); a piece of genomic DNA containing the mouse Polr2a gene promoter (829); a translation initiation sequence (methionine codon embedded in a "Kozak" consensus sequence, 835)); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (831); a transcription termination/polyadenylation sequence (pA. 833); a loxP recognition sequence for the Cre recombinase (837); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphogly cerate kinase 1 gene (839); and 3 Kb of mouse genomic DNA (841) mapping close to the 4.5 Kb mouse genomic DNA sequence present near the 5’ end of the vector and arranged in the native relative orientation.

[000234] The key features of the 3' vector (805) in order from 5' to 3' are as follows; 3.7 Kb of mouse genomic DNA mapping within the intron between the JH and CH gene loci (843); an HPRT gene under transcriptional control of the mouse Polr2a gene promoter (845); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (847); a loxP recognition sequence for the Cre recombinase (837); 2.1 Kb of mouse genomic DNA (849) that maps immediately downstream of the 3.7 Kb mouse genomic DNA fragment present near the 5' end of the vector and arranged in the native relative orientation; and a gene encoding the DTA subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (823).

[000235] Mouse embryonic stem (ES) cells (derived from C57Bl/6NTac mice) are transfected by electroporation with the 3' vector (805) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ~24 hours they are placed under positive selection for cells that have integrated the 3' vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene (823), which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGH locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells can be frozen as an archive, and the rest used for isolation of DNA for analytical purposes.

[000236] DNA from the ES cell clones is screened by PCR using a widely practiced gene targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3' vector (805) and the genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (845) or neomycin resistance (847) genes. According to the standard design, these assays detect pieces of DNA that would only be present in clones of ES cells derived from transfected cells that undergo fully legitimate homologous recombination between the 3' targeting vector and the endogenous mouse IGH locus. Two separate transfections are performed with the 3' vector (805). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.

[000237] The Southern blot assays are performed according to widely used procedures using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allow the structure of the targeted locus in the clones to be identified as properly modified by homologous recombination. One of the probes maps to DNA sequence flanking the 5' side of the region of identity shared between the 3' targeting vector and the genomic DNA; a second probe maps outside the region of identity but on the 3' side; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (845) or neomycin resistance (847) genes. The Southern blot identifies the presence of the expected restriction enzyme generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3' Igh targeting vector, part of the IGH locus as detected by one of the external probes and by the neomycin or HPRT probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin Igh locus on the homologous chromosome.

[000238] Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. ES cell clones that are judged to have the expected correct genomic structure based on the Southern blot data— and that also do not have detectable chromosomal aberrations based on the karyotype analysis— are selected for further use.

[000239] Acceptable clones are then modified with the 5' vector (803) using procedures and screening assays that are similar in design to those used with the 3' vector (805) except that puromycin selection is used instead of G418/neomycin for selection. The PCR assays, probes and digests are also tailored to match the genomic region being modified by the 5' vector (805).

[000240] Clones of ES cells that have been mutated in the expected fashion by both the 3' and the 5' vectors, i.e., doubly targeted cells carrying both engineered mutations, are isolated following vector targeting and analysis. The clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes (i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands). Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (803 and 805) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-TK (839), HPRT (845) and neomycin resistance (847) genes if the targeting vectors have been integrated in cis, and then comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (803) and by analyzing the drug resistance phenotype of the surviving clones by a "sibling selection" screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 10 ³ more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting c/.s-derived ganciclovir-resistant clones are also sensitive to both puromycin and G418/neomycin, in contrast to the trans- derived ganciclovir-resistant clones, which should retain resistance to both drugs. Doubly targeted clones of cells with the cis-arrangement of engineered mutations in the heavy chain locus are selected for further use.

[000241] The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase and the transfected cells subsequently are placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir- resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion between the two engineered mutations created by the 5' (803) and the 3' (805) targeting vectors. In these clones, the Cre recombinase causes a recombination (802) to occur between the loxP sites (837) introduced into the heavy chain locus by the two vectors to create the genomic DNA configuration shown at 807. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and therefore is lost from the cells as they undergo proliferation. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites. Clones that have the expected deletion are selected for further use. [000242] ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin heavy chain locus are retransfected (804) with a Cre recombinase expression vector together with a piece of DNA (809) comprising a partly canine immunoglobulin heavy chain locus containing canine VH, D and JH region gene coding region sequences flanked by mouse regulatory and flanking sequences. The key features of this piece of synthetic DNA (809) are the following: a lox5171 site (831); a neomycin resistance gene open reading frame (847) lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site a FRT site (827); an array of 39 functional canine VH heavy chain variable region genes (851), each with canine coding sequences embedded in mouse noncoding sequences; optionally a 21.6 kb pre-D region from the mouse heavy chain locus (not shown); a 58 Kb piece of DNA containing the 6 canine DH gene segments (853) and 6 canine JH gene segments (855) where the canine VH, D and JH coding sequences are embedded in mouse noncoding sequences; a loxP site (837) in opposite relative orientation to the lox5171 site (831).

[000243] The transfected clones are placed under G418 selection, which enriches for clones of cells that have undergone RMCE in which the engineered partly canine donor immunoglobulin locus (809) is integrated in its entirety into the deleted endogenous immunoglobulin heavy chain locus between the lox5171 (831) and loxP (837) sites to create the DNA region illustrated at 811. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (847) because the promoter (829) as well as the initiator methionine codon (835) required for its expression are not present in the vector (809) but are already pre-existing in the host cell IGH locus (807). The remaining elements from the 5' vector (803) are removed via Flp-mediated recombination (806) in vitro or in vivo , resulting in the final canine-based locus as shown at 813.

[000244] G418-resistant ES cell clones are analyzed by PCR and Southern blot to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.

[000245] ES cell clones carrying the partly canine immunoglobulin heavy chain DNA (813) in the mouse heavy chain locus are microinjected into mouse blastocysts from strain DBA/2 to create partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice here are of C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin heavy chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly canine locus are used to establish a colony of mice.

Example 4: Introduction of an Engineered Partly Canine Immunoglobulin Locus into the Immunoglobulin k Chain Gene Locus of a Mouse Genome

[000246] Another method for replacing a portion of a mouse genome with partly canine immunoglobulin locus is illustrated in FIG. 9. This method includes introducing a first site-specific recombinase recognition sequence into the mouse genome, which may be introduced either 5' or 3' of the cluster of endogenous V _K (915) and J _K (919) region gene segments of the mouse genome, followed by the introduction of a second site-specific recombinase recognition sequence into the mouse genome, which in combination with the first sequence-specific recombination site flanks the entire locus comprising clusters of V _K and J _K gene segments upstream of the constant region gene (921). The flanked region is deleted and then replaced with a partly canine immunoglobulin locus using the relevant site-specific recombinase, as described herein.

[000247] The targeting vectors employed for introducing the site-specific recombination sequences on either side of the V _K (915) and J _K (919) gene segments also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the V _K and J _K gene segment clusters it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified V _K locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising canine V _K and J _K gene segment coding sequences embedded in mouse regulatory and flanking sequences. [000248] Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (903) comprises mouse genomic DNA taken from the 5' end of the locus, upstream of the most distal V _K gene segment. The other vector (905) comprises mouse genomic DNA taken from within the locus downstream (3') of the J _K gene segments (919) and upstream of the constant region genes (921).

[000249] The key features of the 5' vector (903) are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (923); 6 Kb of mouse genomic DNA (925) mapping upstream of the most distal variable region gene in the k chain locus; a FRT recognition sequence for the Flp recombinase (927); a piece of genomic DNA containing the mouse Polr2a gene promoter (929); a translation initiation sequence (935, methionine codon embedded in a "Kozak" consensus sequence); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (931); a transcription termination/polyadenylation sequence (933); a loxP recognition sequence for the Cre recombinase (937); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphoglycerate kinase 1 gene (939); 2.5 Kb of mouse genomic DNA (941) mapping close to the 6 Kb sequence at the 5’ end in the vector and arranged in the native relative orientation.

[000250] The key features of the 3' vector (905) are as follows: 6 Kb of mouse genomic DNA (943) mapping within the intron between the JK (919) and CK (921) gene loci; a gene encoding the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) under transcriptional control of the mouse Polr2a gene promoter (945); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (947); a loxP recognition sequence for the Cre recombinase (937); 3.6 Kb of mouse genomic DNA (949) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5’ end in the vector, with the two fragments oriented in the same relative way as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (923). [000251] Mouse embryonic stem (ES) cells derived from C57B l/6NTac mice are transfected by electroporation with the 3' vector (905) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ~24 hours they are placed under positive selection for cells that have integrated the 3' vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IgK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells could be frozen as an archive, and the rest used for isolation of DNA for analytical purposes.

[000252] DNA from the ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3' vector (905) and the genomic DNA (901), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (945) or neomycin resistance (947) genes. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the 3' vector (905) and the endogenous mouse IgK locus. Two separate transfections are performed with the 3' vector (905). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.

[000253] The Southern blot assays are performed according to widely used procedures; they involve three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. One of the probes maps to DNA sequence flanking the 5' side of the region of identity shared between the 3' k targeting vector (905) and the genomic DNA; a second probe also maps outside the region of identity but on the 3' side; the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (945) or neomycin resistance (947) genes. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3' k targeting vector (905) part of the k locus, as detected by one of the external probes and by the neomycin resistance or HPRT gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin k locus on the homologous chromosome.

[000254] Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyoptypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.

[000255] Acceptable clones are then modified with the 5' vector (903) using procedures and screening assays that are similar in design to those used with the 3' vector (905), except that puromycin selection is used instead of G418/neomycin selection, and the protocols are tailored to match the genomic region modified by the 5' vector (903). The goal of the 5' vector (903) transfection experiments is to isolate clones of ES cells that have been mutated in the expected fashion by both the 3' vector (905) and the 5' vector (903), i.e., doubly targeted cells carrying both engineered mutations. In these clones, the Cre recombinase causes a recombination (902) to occur between the loxP sites introduced into the k locus by the two vectors, resulting in the genomic DNA configuration shown at 907.

[000256] Further, the clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes; i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (903 and 905) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-Tk (939), HPRT (945) and neomycin resistance (947) genes if the targeting vectors have been integrated in cis, and comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (903) and by analyzing the drug resistance phenotype of the surviving clones by a "sibling selection" screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 10 ³ more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting c/.s-derived ganciclovir-resistant clones should also be sensitive to both puromycin and G418/neomycin, in contrast to the trans- derived ganciclovir-resistant clones, which should retain resistance to both drugs. Clones of cells with the c/.s-arrangement of engineered mutations in the k chain locus are selected for further use.

[000257] The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (902) and the transfected cells are subsequently placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion (907) between the two engineered mutations created by the 5' vector (903) and the 3' vector (905). In these clones, the Cre recombinase has caused a recombination to occur between the loxP sites (937) introduced into the k chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites. Clones that have the expected deletion are selected for further use. [000258] The ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin k chain locus are retransfected (904) with a Cre recombinase expression vector together with a piece of DNA (909) comprising a partly canine immunoglobulin k chain locus containing V _K (951) and JK (955) gene segment coding sequences. The key features of this piece of DNA (referred to as "K-K") are the following: a lox5171 site (931); a neomycin resistance gene open reading frame (947, lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (931)); a FRT site (927); an array of 14 canine VK gene segments (951), each with canine coding sequences embedded in mouse noncoding sequences; optionally a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of JK region gene segments in the mouse k chain locus (not shown); a 2 Kb piece of DNA containing the 5 canine JK region gene segments (955) embedded in mouse noncoding DNA; a loxP site (937) in opposite relative orientation to the lox5171 site (931).

[000259] The sequences of the canine VK and JK gene coding regions are in Table 2.

[000260] In a second independent experiment, an alternative piece of partly canine DNA (909) is used in place of the K-K DNA. The key features of this DNA (referred to as "L- K") are the following: a lox5171 site (931); a neomycin resistance gene open reading frame (947) lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (931); a FRT site (927); an array of 76 functional canine V variable region gene segments (951), each with canine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the JK region gene segments in the mouse k chain locus (not shown); a 2 Kb piece of DNA containing 7 canine J region gene segments embedded in mouse noncoding DNA (955); a loxP site (937) in opposite relative orientation to the lox5171 site (931). (The dog has 9 functional h region gene segments, however, the encoded protein sequence of J 4 and J 9 and of J 7 and J 8 are identical, and so only 7 i, gene segments are included.)

[000261] The transfected clones from the K-K and L-K transfection experiments are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly canine donor DNA (909) is integrated in its entirety into the deleted immunoglobulin k chain locus between the lox5171 (931) and loxP (937) sites that were placed there by 5' (903) and 3' (905) vectors, respectively. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (947) because the promoter (929) as well as the initiator methionine codon (935) required for its expression are not present in the vector (909) and are already pre-existing in the host cell Igh locus (907). The DNA region created using the K-K sequence is illustrated at 911. The remaining elements from the 5' vector (903) are removed via Flp-mediated recombination (906) in vitro or in vivo , resulting in the final canine-based light chain locus as shown at 913.

[000262] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Both K-K and L-K clones that have the expected genomic structure are selected for further use.

[000263] The K-K ES cell clones and the L-K ES cell clones carrying the partly canine immunoglobulin DNA in the mouse k chain locus (913) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin k or l light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly canine locus are used to establish colonies of K-K and L-K mice.

[000264] Mice carrying the partly canine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a canine-based xchain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both canine-based loci, i.e., canine-based for heavy chain and K. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine k proteins with canine k variable domains and the mouse K constant domain from their k loci. Monoclonal antibodies recovered from these mice have canine heavy chain variable domains paired with canine k variable domains. [000265] A variation on the breeding scheme involves generating mice that are homozygous for the canine-based heavy chain locus, but heterozygous at the k locus such that on one chromosome they have the K-K canine-based locus and on the other chromosome they have the L-K canine-based locus. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine K proteins with canine k variable domains and the mouse k constant domain from one of their k loci. From the other k locus, they produce partly canine l proteins with canine l variable domains the mouse k constant domain. Monoclonal antibodies recovered from these mice have canine variable domains paired in some cases with canine k variable domains and in other cases with canine l variable domains.

Example 5: Introduction of an Engineered Partly Canine Immunoglobulin Locus into the Immunoglobulin l Chain Gene Locus of a Mouse Genome

[000266] Another method for replacing a portion of a mouse genome with an engineered partly canine immunoglobulin locus is illustrated in FIG. 10. This method comprises deleting approximately 194 Kb of DNA from the wild-type mouse immunoglobulin l locus (1001)— comprising Vxx/Vx 2 gene segments (1013), J , _.2 /07 _.2 gene cluster (1015), and V, _{. i} gene segment (1017)— by a homologous recombination process involving a targeting vector (1003) that shares identity with the locus both upstream of the V, _.x /V,.2 gene segments (1013) and downstream of the V,.i gene segment (1017) in the immediate vicinity of the 1 _l 3, Cx3, Ki and C i l gene cluster (1023). The vector replaces the 194 Kb of DNA with elements designed to permit a subsequent site-specific recombination in which a non native piece of DNA is moved into the modified Vx locus via RMCE (1004). In this example, the non-native DNA is a synthetic nucleic acid comprising both canine and mouse sequences.

[000267] The key features of the gene targeting vector (1003) for accomplishing the 194 Kb deletion are as follows: a negative selection gene such as a gene encoding the A subunit of the diphtheria toxin (DTA, 1059) or a herpes simplex virus thymidine kinase gene (not shown); 4 Kb of genomic DNA from 5' of the mouse Vxx/Vx 2 variable region gene segments in the l locus (1025); a FRT site (1027); a piece of genomic DNA containing the mouse Polr2a gene promoter (1029); a translation initiation sequence (methionine codon embedded in a "Kozak" consensus sequence) (1035); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (1031); a transcription termination/polyadenylation sequence (1033); an open reading frame encoding a protein that confers resistance to puromycin (1037), whereas this open reading frame is on the antisense strand relative to the Polr2a promoter and the translation initiation sequence next to it and is followed by its own transcription termination/polyadenylation sequence (1033); a loxP recognition sequence for the Cre recombinase (1039); a translation initiation sequence (a methionine codon embedded in a "Kozak" consensus sequence) (1035) on the same, antisense strand as the puromycin resistance gene open reading frame; a chicken beta actin promoter and cytomegalovirus early enhancer element (1041) oriented such that it directs transcription of the puromycin resistance open reading frame, with translation initiating at the initiation codon downstream of the loxP site and continuing back through the loxP site into the puromycin open reading frame all on the antisense strand relative to the Polr2a promoter and the translation initiation sequence next to it; a mutated recognition site for the Flp recombinase known as an "F3" site (1043); a piece of genomic DNA upstream of the J ₃ , C 3, hi and Cu gene segments (1045).

[000268] Mouse embryonic stem (ES) cells derived from C57B l/6NTac mice are transfected (1002) by electroporation with the targeting vector (1003) according to widely used procedures. Homologous recombination replaces the native DNA with the sequences from the targeting vector (1003) in the 196 Kb region resulting in the genomic DNA configuration depicted at 1005.

[000269] Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ~24 hours placed under positive drug selection using puromycin. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGL locus. Colonies of drug- resistant ES cells are physically extracted from their plates after they became visible to the naked eye approximately a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells are divided such that some of the cells are frozen as an archive, and the rest used for isolation of DNA for analytical purposes.

[000270] DNA from the ES cell clones is screened by PCR using a widely used gene targeting assay design. For these assays, one of the PCR oligonucleotide primer sequences maps outside the regions of identity shared between the targeting vector and the genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity in the vector, e.g., in the puro gene (1037). According to the standard design, these assays detect pieces of DNA that would only be present in clones of cells derived from transfected cells that had undergone fully legitimate homologous recombination between the targeting vector (1003) and the native DNA (1001).

[000271] Six PCR-positive clones from the transfection (1002) are selected for expansion followed by further analysis using Southern blot assays. The Southern blots involve three probes and genomic DNA from the clones that has been digested with multiple restriction enzymes chosen so that the combination of probes and digests allow identification of whether the ES cell DNA has been properly modified by homologous recombination.

[000272] Karyotypes of the six PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most common chromosomal aberrations that arise in mouse ES cells. Clones that show evidence of aberrations are excluded from further use. Karyoptypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.

[000273] The ES cell clones carrying the deletion in one of the two homologous copies of their immunoglobulin l chain locus are retransfected (1004) with a Cre recombinase expression vector together with a piece of DNA (1007) comprising a partly canine immunoglobulin l chain locus containing V>„ J> _. and C,_ region gene segments. The key features of this piece of DNA (1007) are as follows: a lox5171 site (1031); a neomycin resistance gene open reading frame lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1047); a FRT site 1027); an array of 76 functional canine l region gene segments, each with canine l coding sequences embedded in mouse l noncoding sequences (1051); an array of J-C units where each unit has a canine Jx gene segment and a mouse l constant domain gene segment embedded within noncoding sequences from the mouse l locus (1055) (the canine Jx gene segments are those encoding Jxi, Jx2, J,j, Jx4, J , Jxr _> , and Jx?, while the mouse l constant domain gene segments are C i or 0 _.2 or CO); a mutated recognition site for the Flp recombinase known as an "F3" site (1043); an open reading frame conferring hygromycin resistance (1057), which is located on the antisense strand relative to the immunoglobulin gene segment coding information in the construct; a loxP site (1039) in opposite relative orientation to the lox5171 site.

[000274] The sequences of the canine Vx and Jx gene coding regions are in Table 3.

[000275] The transfected clones are placed under G418 or hygromycin selection, which enriches for clones of cells that have undergone a RMCE process, in which the partly canine donor DNA is integrated in its entirety into the deleted immunoglobulin l chain locus between the lox5171 and loxP sites that were placed there by the gene targeting vector. The remaining elements from the targeting vector (1003) are removed via FLP- mediated recombination (1006) in vitro or in vivo resulting in the final caninized locus as shown at 1011.

[000276] G418/hygromycin-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected recombinase-mediated cassette exchange process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.

[000277] The ES cell clones carrying the partly canine immunoglobulin DNA (1011) in the mouse l chain locus are microinjected into mouse blastocysts from strain DBA/2 to create partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice here are of the C57Bl/6NTac strain, which carry a transgene encoding the Flp recombinase expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin l chain locus, and for loss of the FRT-flanked neomycin resistance gene and the F3-flanked hygromycin resistance gene that were created in the RMCE step. Mice that carry the partly canine locus are used to establish a colony of mice. [000278] In some aspects, the mice comprising the canine-based heavy chain and k locus (as described in Examples 3 and 4) are bred to mice that carry the canine-based l locus. Mice generated from this type of breeding scheme are homozygous for the canine-based heavy chain locus, and can be homozygous for the K-K canine-based locus or the L-K canine- based locus. Alternatively, they can be heterozygous at the k locus carrying the K-K locus on one chromosome and the L-K locus on the other chromosome. Each of these mouse strains is homozygous for the canine-based l locus. Monoclonal antibodies recovered from these mice has canine heavy chain variable domains paired in some cases with canine K variable domains and in other cases with canine l variable domains. The l variable domains are derived from either the canine-based L-K locus or the canine-based l locus.

Example 6: Introduction of an Engineered Partly Canine Immunoglobulin Minilocus into a Mouse Genome

[000279] In certain other aspects, the partly canine immunoglobulin locus comprises a canine variable domain minilocus such as the one illustrated in FIG. 11. Here instead of a partly canine immunoglobulin locus comprising all or substantially all of the canine VH gene segment coding sequences, the mouse immunoglobulin locus is replaced with a minilocus (1119) comprising fewer chimeric canine VH gene segments, e.g. 1-39 canine VH gene segments determined to be functional; that is, not pseudogenes.

[000280] A site-specific targeting vector (1131) comprising the partly canine immunoglobulin locus to be integrated into the mammalian host genome is introduced (1102) into the genomic region (1101) with the deleted endogenous immunoglobulin locus comprising the puro-TK gene (1105) and the following flanking sequence-specific recombination sites: mutant FRT site (1109), mutant LoxP site (1111), wild-type FRT site (1107), and wild-type LoxP site (1105). The site-specific targeting vector comprises i) an array of optional PAIR elements (1141); ii) a VH locus (1119) comprising, e.g., 1-39 functional canine VH coding regions and intervening sequences based on the mouse genome endogenous sequences; iii) a 21.6 kb pre-D region (1121) comprising mouse sequence; iv) a D locus (1123) and a JH locus (1125) comprising 6 D and 6 JH canine coding sequences and intervening sequences based on the mouse genome endogenous sequences. The partly canine immunoglobulin locus is flanked by recombination sites— mutant FRT (1109), mutant LoxP (1111), wild-type FRT (1107), and wild-type LoxP (1105)— that allow recombination with the modified endogenous locus. Upon introduction of the appropriate recombinase, e.g., Cre) (1104), the partly canine immunoglobulin locus is integrated into the genome upstream of the constant gene region (1127) as shown at 1129.

[000281] As described in Example 1, the primary screening for introduction of the partly canine immunoglobulin variable region locus is carried out by primary PCR screens supported by secondary Southern blotting assays. The deletion of the puro-TK gene (1105) as part of the recombination event allows identification of the cells that did not undergo the recombination event using ganciclovir negative selection.

Example 7: Introduction of an Engineered Partly Canine Immunoglobulin Locus with Canine l Variable Region Coding Sequences with Mouse l Constant Region Sequences embedded in k Immunoglobulin Non-coding Sequences

[000282] Dog antibodies mostly contain l light chains, whereas mouse antibodies mostly contain k light chains. To increase production of antibodies containing a l LC, the endogenous mouse V _K and J _K are replaced with a partly canine locus containing V, _. and U gene segment coding sequences embedded in mouse VK region flanking and regulatory sequences, the L-K mouse of Example 4. In such a mouse, the endogenous regulatory sequences promoting high level k locus rearrangement and expression are predicted to have an equivalent effect on the ectopic l locus. However, in vitro studies demonstrated that canine Vx domains do not function well with mouse C _K (see Example 9). Thus, the expected increase in l LC-containing antibodies in the L-K mouse might not occur. As an alternate strategy, the endogenous mouse V _K and J _K are replaced with a partly canine locus containing V and J, _. gene segment coding sequences embedded in mouse V _K region flanking and regulatory sequences and mouse C _K is replaced with mouse Cx.

[000283] FIG. 13 is a schematic diagram illustrating the introduction of an engineered partly canine light chain variable region locus in which one or more canine V gene segment coding sequences are inserted into a rodent immunoglobulin k light chain locus upstream of one or more canine Jx gene segment coding sequences, which are upstream of one or more rodent Cx region coding sequences. [000284] The method for replacing a portion of a mouse genome with a partly canine immunoglobulin locus is illustrated in FIG. 13. This method includes introducing a first site-specific recombinase recognition sequence into the mouse genome, which may be introduced either 5' or 3' of the cluster of endogenous VK (1315) and JK (1319) region gene segments and the CK (1321) exon of the mouse genome, followed by the introduction of a second site-specific recombinase recognition sequence into the mouse genome, which in combination with the first sequence-specific recombination site flanks the entire locus comprising clusters of VK and JK gene segments and the CK exon. The flanked region is deleted and then replaced with a partly canine immunoglobulin locus using the relevant site-specific recombinase, as described herein.

[000285] The targeting vectors employed for introducing the site-specific recombination sequences on either side of the VK (1315) gene segments and the CK exon (1321) also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the V _K and JK gene segment clusters and the CK exon it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified VK locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprises canine V and J gene segment coding sequences and mouse Cx exon(s) embedded in mouse IGK regulatory and flanking sequences.

[000286] Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (1303) comprises mouse genomic DNA taken from the 5' end of the locus, upstream of the most distal VK gene segment. The other vector (1305) comprises mouse genomic DNA taken from within the locus in a region spanning upstream (5’) and downstream (3') of the CK exon (1321).

[000287] The key features of the 5' vector (1303) are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1323); 6 Kb of mouse genomic DNA (1325) mapping upstream of the most distal variable region gene in the k chain locus; a FRT recognition sequence for the Flp recombinase (1327); a piece of genomic DNA containing the mouse Polr2a gene promoter (1329); a translation initiation sequence (1335, methionine codon embedded in a "Kozak" consensus sequence); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (1331); a transcription termination/polyadenylation sequence (1333); a loxP recognition sequence for the Cre recombinase (1337); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphogly cerate kinase 1 gene (1339); 2.5 Kb of mouse genomic DNA (1341) mapping close to the 6 Kb sequence at the 5’ end in the vector and arranged in the native relative orientation.

[000288] The key features of the 3' vector (1305) are as follows: 6 Kb of mouse genomic DNA (1343) mapping within the locus in a region spanning upstream (5’) and downstream (3') of the C _K exon (1321); a gene encoding the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) under transcriptional control of the mouse Polr2a gene promoter (1345); a neomycin resistance gene under the control of the mouse phosphogly cerate kinase 1 gene promoter (1347); a loxP recognition sequence for the Cre recombinase (1337); 3.6 Kb of mouse genomic DNA (1349) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5’ end in the vector, with the two fragments oriented in the same relative way as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1323).

[000289] One strategy to delete the endogenous mouse IGK locus is to insert the 3' vector (1305) in the flanking region downstream of the mouse C _K exon (1321). However, the 3’K enhancer, which needs to be retained in the modified locus, is located 9.1 Kb downstream of the C _K exon, which is too short to accommodate the upstream and downstream homology arms of the 3’ vector, which total 9.6 Kb. Therefore, the upstream region of homology was extended.

[000290] Mouse embryonic stem (ES) cells derived from C57B l/6NTac mice are transfected by electroporation with the 3' vector (1305) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ~24 hours they are placed under positive selection for cells that have integrated the 3' vector into their DNA using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination retains the DTA gene, which kills the cells when the gene is expressed, but the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IgK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they are visible to the naked eye about a week later. These colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided - some of the cells are frozen as an archive, and the rest are used to isolate DNA for analytical purposes.

[000291] DNA from the ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3' vector (1305) and the genomic DNA (1301), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (1345) or neomycin resistance (1347) genes. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the 3' vector (1305) and the endogenous mouse IgK locus. Two separate transfections are performed with the 3' vector (1305). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.

[000292] Southern blot assays are performed according to widely used procedures using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. A first probe maps to DNA sequence flanking the 5' side of the region of identity shared between the 3' k targeting vector (1305) and the genomic DNA; a second probe also maps outside the region of identity but on the 3' side; a third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (1345) or neomycin resistance (1347) genes. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3' k targeting vector (1305) part of the K locus, as detected by one of the external probes and by the neomycin resistance or HPRT gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin k locus on the homologous chromosome.

[000293] Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyoptypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.

[000294] Acceptable clones are then modified with the 5' vector (1303) using procedures and screening assays that are similar in design to those used with the 3' vector (1305), except that puromycin selection is used instead of G418/neomycin selection, and the protocols are tailored to match the genomic region modified by the 5' vector (1303). The goal of the 5' vector (1303) transfection experiments is to isolate clones of ES cells that have been mutated in the expected fashion by both the 3' vector (1305) and the 5' vector (1303), i.e., doubly targeted cells carrying both engineered mutations. In these clones, the Cre recombinase causes a recombination (1302) to occur between the loxP sites introduced into the K locus by the two vectors, resulting in the genomic DNA configuration shown at 1307.

[000295] Further, the clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes; i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (1303 and 1305) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-Tk (1339), HPRT (1345) and neomycin resistance (1347) genes if the targeting vectors have been integrated in cis , and comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (1303) and by analyzing the drug resistance phenotype of the surviving clones by a "sibling selection" screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 10 ³ more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting c/.s-derived ganciclovir-resistant clones should also be sensitive to both puromycin and G418/neomycin, in contrast to the trans- derived ganciclovir-resistant clones, which should retain resistance to both drugs. Clones of cells with the c/.s-arrangement of engineered mutations in the k chain locus are selected for further use.

[000296] The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (1302) and the transfected cells are subsequently placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion (1307) between the two engineered mutations created by the 5' vector (1303) and the 3' vector (1305). In these clones, the Cre recombinase causes a recombination to occur between the loxP sites (1337) introduced into the K chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites and have the genomic structure show at 1307. Clones that have the expected deletion are selected for further use.

[000297] The ES cell clones carrying the sequence deletion in one of the two homologous copies of their immunoglobulin k chain locus are retransfected (1304) with a Cre recombinase expression vector together with a piece of DNA (1309) comprising a partly canine immunoglobulin l chain locus containing V (1351) and T (1355) gene segment coding sequences and mouse Cx exon(s) (1357). The key features of this piece of DNA are the following: alox5171 site (1331); a neomycin resistance gene open reading frame (1347, lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1331); a FRT site (1327); an array of 1-76 functional canine V variable region gene segments (1351), each with canine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the JK region gene segments in the mouse k chain locus (not shown); a 2 Kb piece of DNA containing 1-7 canine J region gene segments embedded in mouse noncoding DNA (1355) and mouse C exon(s) (1357); a loxP site (1337) in opposite relative orientation to the lox5171 site (1331). The piece of DNA also contains the deleted ΐEk (not shown).

[000298] The sequences of the canine V and J gene coding regions are in Table 3.

[000299] The transfected cells are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly canine donor DNA (1309) is integrated in its entirety into the deleted immunoglobulin k chain locus between the lox5171 (1331) and loxP (1337) sites that were placed there by 5' (1303) and 3' (1305) vectors, respectively. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (1347) because the promoter (1329) as well as the initiator methionine codon (1335) required for its expression are not present in the vector (1309) and are already pre-existing in the host cell IGK locus (1307). The DNA region created by RMCE is illustrated at 1311. The remaining elements from the 5' vector (1303) are removed via Flp-mediated recombination (1306) in vitro or in vivo , resulting in the final canine-based light chain locus as shown at 1313.

[000300] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.

[000301] Clones carrying the partly canine immunoglobulin DNA in the mouse k chain locus (1313) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell- derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin l light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly canine locus are used to establish colonies of mice.

[000302] Mice carrying the partly canine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a canine l-based k chain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both canine-based loci, i.e., canine-based for heavy chain and l-based l. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine l proteins with canine l variable domains and the mouse l constant domain from their k loci. Monoclonal antibodies recovered from these mice have canine heavy chain variable domains paired with canine l variable domains.

[000303] A variation on the breeding scheme involves generating mice that are homozygous for the canine-based heavy chain locus, but heterozygous at the k locus such that on one chromosome they have the K-K canine-based locus described in Example 4 and on the other chromosome they have the partly canine l-based k locus described in this example. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine k proteins with canine k variable domains and the mouse k constant domain from one of their k loci. From the other k locus, partly canine l proteins comprising canine l variable domains and the mouse l constant domain are produced. Monoclonal antibodies recovered from these mice include canine variable domains paired in some cases with canine k variable domains and in other cases with canine l variable domains.

Example 8 Introduction of an Engineered Partly Canine Immunoglobulin Locus with Canine l Variable Region Coding Sequences with Mouse l Constant Region Sequences embedded in Mouse k Immunoglobulin Non-coding Sequences

[000304] This example describes an alternate strategy to Example 7 in which the endogenous mouse V _K and J _K are replaced with a partly canine locus containing canine U _l and J> _. gene segment coding sequences embedded in mouse VK region flanking and regulatory sequences and mouse CK is replaced with mouse Cx. However, in this example the structure of the targeting vector containing the partly canine locus is different. The canine V gene locus coding sequences include an array of anywhere from 1 to 76 functional V, _. gene segment coding sequences, followed by an array of Jx-Cx tandem cassettes in which the Jx is of canine origin and the Cx is of mouse origin, for example, C i, Cx2 or Cx _3. The number of cassettes ranges from one to seven, the number of unique functional canine Jx gene segments. The overall structure of the partly canine l locus in this example is similar to the endogenous mouse l locus, whereas the structure of the locus in Example 7 is similar to the endogenous mouse k locus, which is being replaced by the partly canine l locus in that example.

[000305] FIG. 14 is a schematic diagram illustrating the introduction of an engineered partly canine light chain variable region locus in which one or more canine Vx gene segment coding sequences are inserted into a rodent immunoglobulin k light chain locus upstream of an array of Jx-Cx tandem cassettes in which the Jx is of canine origin and the Cx is of mouse origin, for example, Cxi, Cx2 or Cx3.

[000306] The method for replacing a portion of a mouse genome with a partly canine immunoglobulin locus is illustrated in FIG. 14. This method provides introducing a first site-specific recombinase recognition sequence into the mouse genome, which may be introduced either 5' or 3' of the cluster of endogenous V _K (1415) and J _K (1419) region gene segments and the C _K (1421) exon of the mouse genome, followed by the introduction of a second site-specific recombinase recognition sequence into the mouse genome, which in combination with the first sequence-specific recombination site flanks the entire locus comprising clusters of V _K and J _K gene segments and the C _K exon. The flanked region is deleted and then replaced with a partly canine immunoglobulin locus using the relevant site-specific recombinase, as described herein.

[000307] The targeting vectors employed for introducing the site-specific recombination sequences on either side of the V _K (1415) gene segments and the C _K exon (1421) also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the V _K and J _K gene segment clusters and the C _K exon it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified V _K locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising an array of canine Vx gene segment coding sequences and an array of T- C . tandem cassettes in which the T is of canine origin and the Cx is of mouse origin, for example, C i, 0.2 or G _.i embedded in mouse IGK regulatory and flanking sequences.

[000308] Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (1403) comprises mouse genomic DNA taken from the 5' end of the locus, upstream of the most distal V _K gene segment. The other vector (1405) comprises mouse genomic DNA taken from within the locus in a region spanning upstream (5’) and downstream (3') of the C _K exon (1321).

[000309] The key features of the 5' vector (1403) and the 3' vector (1405) are described in Example 7.

[000310] Mouse embryonic stem (ES) cells derived from C57B l/6NTac mice are transfected by electroporation with the 3' vector (1405) according to widely used procedures as described in Example 7. DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay as described in Example 7. The Southern blot assays are performed according to widely used procedures as described in Example 7.

[000311] Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyoptypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.

[000312] Acceptable clones are modified with the 5' vector (1403) using procedures and screening assays as described in Example 7. The resulting correctly targeted ES clones have the genomic DNA configuration of the endogenous k locus in which the 5’ vector (1403) is inserted upstream of endogenous V _K gene segments and the 3’ vector (1405) is inserted downstream of the endogenous C _K . In these clones, the Cre recombinase causes recombination (1402) to occur between the loxP sites introduced into the k locus by the two vectors, resulting in the genomic DNA configuration shown at 1407.

[000313] Acceptable clones undergo gene targeting on the same chromosome, as opposed to homologous chromosomes; such that the engineered mutations created by the targeting vectors are in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures as described in Example 7.

[000314] The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (1402) and the transfected cells are subsequently placed under ganciclovir selection and analyses using procedures described in Example 7. In selected clones, the Cre recombinase has caused a recombination to occur between the loxP sites (1437) introduced into the k chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites and have the genomic structure show at 1407. Clones that have the expected deletion are selected for further use.

[000315] The ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin k chain locus are retransfected (1404) with a Cre recombinase expression vector together with a piece of DNA (1409) comprising a partly canine immunoglobulin l chain locus containing V (1451) segment coding sequences and a tandem array of cassettes containing canine T gene segment coding sequences and mouse C exon(s) embedded in mouse IGK flanking and regulatory DNA sequences (1457). The key features of this piece of DNA are the following: a lox5171 site (1431); a neomycin resistance gene open reading frame (1447, lacking the initiator methionine codon, but in- frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1431); a FRT site (1427); an array of 1-76 functional canine V variable region gene segments (1451), each containing canine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the J _K region gene segments in the mouse k chain locus (not shown); DNA containing a tandem array of cassettes containing canine T gene segment coding sequences and mouse Cx exon(s) embedded in mouse IGK flanking and regulatory DNA sequences (1457); a loxP site (1437) in opposite relative orientation to the lox5171 site (1431).

[000316] The sequences of the canine V and J gene coding regions are in Table 3.

[000317] The transfected cells are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly canine donor DNA (1409) is integrated in its entirety into the deleted immunoglobulin k chain locus between the lox5171 (1431) and loxP (1437) sites placed there by the 5' (1403) and 3' (1405) vectors, respectively. Only cells that properly undergo RMCE have the capability to express the neomycin resistance gene (1447) because the promoter (1429) as well as the initiator methionine codon (1435) required for its expression are not present in the vector (1409) and are already pre-existing in the host cell IGK locus (1407). The DNA region created by RMCE is illustrated at 1411. The remaining elements from the 5' vector (1403) are removed via Flp-mediated recombination (1406) in vitro or in vivo , resulting in the final canine-based light chain locus as shown at 1413.

[000318] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.

[000319] Clones carrying the partly canine immunoglobulin DNA in the mouse k chain locus (1413) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell- derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin l light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly canine locus are used to establish colonies of mice. [000320] Mice carrying the partly canine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a canine l-based k chain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both canine-based loci, i.e., canine-based for heavy chain and l-based K. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine l proteins with canine l variable domains and the mouse l constant domain from their k loci. Monoclonal antibodies recovered from these mice have canine heavy chain variable domains paired with canine l variable domains.

[000321] A variation on the breeding scheme involves generating mice that are homozygous for the canine-based heavy chain locus, but heterozygous at the k locus such that on one chromosome they have the K-K canine-based locus described in Example 4 and on the other chromosome they have the partly canine l-based k locus described in this example. Such mice produce partly canine heavy chains with canine variable domains and mouse constant domains. They also produce partly canine k proteins with canine k variable domains and the mouse k constant domain from one of their k loci. From the other k locus, they produce partly canine l proteins with canine l variable domains and the mouse l constant domain. Monoclonal antibodies recovered from these mice have canine variable domains paired in some cases with canine k variable domains and in other cases with canine l variable domains.

[000322] The method described above for introducing an engineered partly canine immunoglobulin locus with canine l variable region coding sequences and mouse l constant region sequences embedded in mouse k immunoglobulin non-coding sequences involve deletion of the mouse C _K exon. An alternate method involves inactivating the C _K exon by mutating its splice acceptor site. Introns must be removed from primary mRNA transcripts by a process known as RNA splicing in which the spliceosome, a large molecular machine located in the nucleus, recognizes sequences at the 5’ (splice donor) and 3’ (splice acceptor) ends of the intron, as well as other features of the intron including a polypyrimidine tract located just upstream of the splice acceptor. The splice donor sequence in the DNA is NGT, where“N” is any deoxynucleotide and the splice acceptor is AGN (Cech TR, Steitz JA and Atkins JF Eds. (2019) (RNA Worlds: New Tools for Deep Exploration, CSHL Press) ISBN 978-1-621822-24-0).

[000323] The mouse C _K exon is inactivated by mutating its splice acceptor sequence and the polypyrimidine tract. The wild type sequence upstream of the C _K exon is CTTCCTTCCTCAG (SEQ ID NO: 470) (the splice acceptor site is underlined). It is mutated to AAATTAATTAACC (SEQ ID NO: 471), resulting in a non-functional splice acceptor site and thus a non-functional C _K exon. The mutant sequence also introduces a Pad restriction enzyme site (underlined). As an eight base pair recognition sequence, this restriction site is expected to be present only rarely in the mouse genome (~ every 65,000 bp), making it simple to detect whether the mutant sequence has been inserted into the IGK locus by Southern blot analysis of the ES cell DNA that has been digested with Pad and another, more frequently cutting restriction enzyme. The wild type sequence is replaced with the mutant sequence by homologous recombination, a technique widely known in the art, as to insert the 3’ RMCE vector. The key features of the homologous recombination vector (MSA, 1457) to mutate the C _K exon splice acceptor sequence and the polypyrimidine tract are as follows: 6 Kb of mouse genomic DNA (1443) mapping within the k locus in a region spanning upstream (5’) and downstream (3') of the C _K exon (1421) and containing the mutant AAATTAATTAACC (SEQ ID NO: 471) (1459) sequence instead of the wild type CTTCCTTCCTCAG (SEQ ID NO: 470) sequence in its natural position just upstream of the C _K exon; a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (1447) and flanked by mutant FRT sites (1461); 3.6 Kb of mouse genomic DNA (1449) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5’ end in the vector, with the two fragments oriented in the same relative way as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1423). Mutant FRT sites (1461), e.g., FRT F3 or FRT F5 (Schlake and Bode (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33: 12746-12751 PMID: 7947678 DOI: 10.1021/bi00209a003), are being used here because, once the spicing mutation is introduced and the Neo gene is deleted by transient transfection of a FLP recombinase expression vector (1406), the ES cells are subjected to further genetic manipulation. This process requires wild type FRT sites to delete another Neo selection gene (1447 at 1403). If the FRT site (1461) remaining in the IGK locus (1469) after introduction of the splicing mutation is wild type, attempted FRT-mediated deletion of this second Neo gene (1406 at 1413) may inadvertently result in deletion of the entire newly- introduced partly canine locus and the inactivated mouse C _K exon.

[000324] Mouse embryonic stem (ES) cells derived from C57B l/6NTac mice are transfected by electroporation with the MSA vector (1457) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ~24 hours they are placed under positive selection for cells that have integrated the MSA vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells are frozen as an archive, and the rest used to isolate DNA for analytical purposes.

[000325] The IGK locus in ES cells that are correctly targeted by homologous recombination has the configuration depicted at 1463.

[000326] DNA from the ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the MSA vector (1457) and the genomic DNA (1401), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the neomycin resistance (1447) gene. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the MSA vector (1457) and the endogenous mouse IGK locus. Two separate transfections are performed with the MSA vector (1457). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.

[000327] The Southern blot assays are performed according to widely used procedure using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. In in this particular example, the DNA is double digested with Pad and another restriction enzyme such as EcoRI or Hindlll, as only cells with the integrated MSA vector contains the Pad site. A first probe maps to DNA sequence flanking the 5' side of the region of identity shared between the MSA vector (1457) and the genomic DNA; a second probe also maps outside the region of identity but on the 3' side; a third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the neomycin resistance (1447) gene. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the MSA k targeting vector (1457) part of the K locus, as detected by one of the external probes and by the neomycin resistance gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin k locus on the homologous chromosome. The Southern blot assays are performed according to widely used procedures described in Example 7.

[000328] Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyoptypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.

[000329] Although the ability of the ES cell DNA to be digested by Pad in the mutated IGK allele confirms the presence of the TTAATTAA sequence, DNA sequencing focusing on the region upstream of the C _K exon is performed to confirm the presence of the complete expected splicing mutation. The region is amplified by genomic PCR using primers that flank the mutation [1465 and 1467 (Table 6: SEQ ID NO: 417 and SEQ ID NO:418)]. An alternate primer pair is shown in SEQ ID NO: 419 and SEQ ID NO: 420. These primers are designed using NCBI Primer-Blast and verified in silico to lack any predicted off-target binding sites in the mouse genome.

[000330] Sequence-verified ES cell clones are transiently transfected (1406) with a FLP recombinase expression vector to delete the neomycin resistance gene (1427). The cells are then subcloned and the deletion is confirmed by PCR. The IGK locus in the ES cells have the genomic configuration depicted at 1469.

[000331] The ES cells are electroporated with the 5’ and 3’ RMCE vectors, as described above. The only differences are that the 3’ vector (1405) is inserted upstream of the mutant C _K exon at the position shown in FIG. 9 at 901 and upstream and downstream homology arms of the 3’ vector (1405) is replaced by the sequences 943 and 949, respectively of the 3’ vector (905) shown in FIG. 9. As a result, PCR primers and Southern blot probes used to test for correct integration of the 3’ vector (1405) are derived from sequences 943 and 949 instead of 1443 and 1449. The ΐEk enhancer is not included in the targeting vector (1409), since this sequence was not deleted.

Example 9: Canine Ul domains do not function well with mouse CK domains and canine VK domains do not function well with mouse CX domains.

[000332] For the proposed L-K mouse (Example 4), canine V and J gene segment coding sequences flanked by mouse non-coding and regulatory sequences are embedded in the mouse IGK locus from which endogenous V _K and J _K gene segments have been deleted. After productive V J gene rearrangement, the resulting Ig gene encodes a LC with a canine l variable domain and a mouse k constant domain. To test whether such a hybrid LC was properly expressed and forms an intact Ig molecule, a series of transient transfection assays were performed with different combinations of Vs, both V _K and V , and C light chain exons, both C _K and C , together with an Ig HC and tested for cell surface and intracellular expression and secretion of the encoded Ig. [000333] For these experiments canine IGHV3-5 (Accession No. MF785020.1), IGHV3-19 (Accession No. FJ197781.1) or IGHV4-1 (Accession No. DN362337.1) linked to a mouse IgM ^b allotype HC was individually cloned into a pCMV vector. Each VH-encoding DNA contained the endogenous canine Ll-intron-L2 and germline, i.e., unmutated VDJ sequence. Unmutated canine IGLV3-28 (Accession No. EU305423) or IGKV2-5 (Accession No. EU295719.1) were cloned into a pFUSE vector. Each canine VL exon was linked to the constant region of mouse C _K , Cu or Cu (C 3 was presumed to have the same properties as Cu since they have nearly identical protein sequence.) Ll-intron-L2 sequences in each VL were of canine origin. 293T/17 cells were co-transfected with a human CD4 expression vector as a transfection control plus one of the HC and LC constructs and a CD79a/b expression vector. The CD79a/b heterodimer was required for cell surface expression of the IgM. Approximately 24h later, the transfected cells were subjected to cell surface or intracellular staining by flow cytometry. For analysis of Ig secretion the same VH genes as above were cloned into a pFUSE vector containing mouse IgG2a Fc. 293T/17 cells were co-transfected with a human CD4 (hCD4) expression vector as a transfection control plus one of the HC and LC constructs described above. (In these experiments C 3 was also tested.) Approximately 48hr later, the transfected cells and their corresponding supernatants were harvested and analyzed for HC/LC expression/secretion by western blotting.

[000334] To summarize the data obtained from these experiments, when canine IGLV3-28 was linked to mouse C _K , IgM expression on the cell surface was at least two times less than when the same dog V was linked to Cu or Cu. Likewise, when IGKV2-5 was linked to mouse C , the level of surface IgM was drastically decreased. The extent of the expression defect was dependent of the particular VH gene being used; some VH genes allowed for some cell surface expression of the hybrid light chains, but others were more stringent. The same trends were seen with Ig secretion.

[000335] FIG. 15 shows the results of flow cytometry analysis of cells expressing IGHV3-5, which was one of the less stringent VH genes, with canine IGVL3-28/IGLJ6 (1501) or with canine IGVK2-5/IGJK1 (1502). The top row panels are transfection controls stained with hCD4 mAh antibody (1509) and the bottom panels were stained with mouse IgM ^b allotype mAh (1510). The non-transfected, hCD4- cells (1513) and transfected, hCD4+ cells (1514) are indicated in all panels by the different shaded histograms. The frequency of non- transfected, hCD4- cells is indicated by the number in the upper left of each panel in the top row and the frequency of transfected, hCD4+ cells is indicated by the number in the upper right of each panel in the top row. Transfection efficiency was similar in all cases. However, when canine V was linked to mouse C _K (1503, bottom row) IgM expression on the cell surface was less than when the same canine V was linked to mouse Cu or Cu (1504, 1505, bottom row) Similarly, the canine IgM with V _K was expressed better when linked to C _K (1506, bottom row) than to Cu or Cu (1507, 1508, bottom row). The numbers in the upper right of each panel in the bottom row indicate the mean fluorescence intensity (MFI) of the cell surface IgM ^b staining, which is a quantitative indication of the level of expression.

[000336] FIG. 16 shows the results of flow cytometry analysis of cells expressing IGHV3-5, which was one of the less stringent VH genes, with canine IGVL3-28/IGLJ6 (1601) or with canine IGVK2-5/IGJK1 (1602). These were the same cells as in FIG. 15, but were stained for cell surface mouse k LC (1609) or mouse l LC (1610), confirming the results shown in FIG. 15.

[000337] FIG. 17 shows the results of flow cytometry analysis of cells expressing IGHV4-1, which was more stringent than IGHV3-5, with canine IGVL3-28/IGLJ6 (1701) or with canine IGVK2-5/IGJK1 (1702). The top row panels are transfection controls stained with hCD4 mAb antibody (1709) and the bottom panels are stained with mouse IgM ^b allotype mAb (1710). The non-transfected, hCD4- cells (1713) and transfected, hCD4+ cells (1714) are indicated in all lower panels by the different shaded histograms. The frequency of non- transfected, hCD4- cells is indicated by the number in the upper left of each panel in the top row and the frequency of transfected, hCD4+ cells is indicated by the number in the upper right of each panel in the top row. Transfection efficiency was similar in all cases. However, when canine V was linked to mouse C _K (1703, bottom row) IgM expression on the cell surface was much less than when the same canine V was linked to mouse Cu or C 2 (1704, 1705, bottom row), although the best expression in this case was with Cu (1705, bottom row). Similarly, the canine IgM with V _K was expressed much better when linked to C _K (1706, bottom row) than to Cu or Cu (1707, 1708, bottom row). In fact, in this case, expression of IgM with Cu or Cu was essentially undetectable. The numbers in the upper right of each panel in the bottom row indicate the mean fluorescence intensity (MFI) of the cell surface IgM ^b staining, which is a quantitative indication of the level of expression. Staining with antibodies specific for mouse l LC or k LC was performed in all experiments and confirmed the results of staining with the IgM ^b allotype mAh (not shown).

[000338] FIG. 18 shows the results of flow cytometry analysis of cells expressing IGHV3- 19, which was the most stringent of the IGHV genes tested in terms of the ability of canine V, _. to function with mouse C _K , with canine IGVL3-28/IGLJ6 (1801) or with canine IGVK2- 5/IGJK1 (1802). The top row panels are transfection controls stained with hCD4 mAh antibody (1809) and the bottom panels are stained with mouse IgM ^b allotype mAh (1810). The non-transfected, hCD4- cells (1813) and transfected, hCD4+ cells (1814) are indicated in all lower panels by the different shaded histograms. The frequency of non-transfected, hCD4- cells is indicated by the number in the upper left of each panel in the top row and the frequency of transfected, hCD4+ cells is indicated by the number in the upper right of each panel in the top row. Transfection efficiency was similar in all cases. There was essentially no surface IgM expression when the canine V was linked to mouse C _K (1803, bottom row) and only low-level expression when the canine V _K was linked to mouse C.i or C 2 (1807, 1808, bottom row). The numbers in the upper right of each panel in the bottom row indicate the mean fluorescence intensity (MFI) of the cell surface IgM ^b staining, which is a quantitative indication of the level of expression. Staining with antibodies specific for mouse l LC or k LC was performed in all experiments and confirmed the results of staining with the IgM ^b allotype mAh (not shown).

[000339] The results of this analysis indicate that hybrid light chains that include canine V and mouse C _K or canine V _K and mouse Cu or C.2 were often poorly expressed on the cell surface with pHC. The level of cell surface IgM was dependent on the particular VH used by the pHC, but there was no discemable pattern that would allow prediction of whether a particular VH would allow modest or no cell surface IgM expression. Since B cell survival depends on IgM BCR expression, pairing of canine V and mouse C _K would result in a major reduction in the development of kLC-expressing B cells. Similarly, pairing of canine V _K with mouse Ci or C.2 would reduce the development of K-LC expressing B cells.

[000340] Expression and secretion of the Ig with hybrid or homologous LC was also tested. Supernatants and cell lysates of the transiently transfected cells were analyzed by western blotting. FIG. 19A shows the results of supernatants of cells using canine IGVL3-28 paired with mouse C _K , C.i, 0.2 or Cx ₃ and a mouse IgG2a HC containing canine IGHVH3-5 (1901), IGHVH3-19 (1902) or IGHVH4-1 (1903). FIG. 19B shows the results of lysates of cells using canine IGVL3-28 paired with mouse C _K , Cxi, Cx2 or Cx ₃ and a mouse IgG2a HC containing canine IGHVH3-5 (1904), IGHVH3-19 (1905) or IGHVH4-1 (1906). The samples were electrophoresed under non-reducing (not shown) or reducing conditions and the blot was probed with an IgG2a antibody. The amount of IgG2a secreted when canine IGVL3-28 was paired with mouse C _K (1907) was consistently much less than when it was paired with Cxi (1908) Cx2 (1909) or Cx ₃ (1910) (FIG. 18A). This difference was not due to lower expression or enhanced degradation of the y2a HC in the canine IGVL3 -28-mouse C _K cells, since the levels were similar in each group of the transfectants (FIG. 19B), or to less protein being analyzed. Loading controls, Myc (FIG. 20A) and GAPDH (FIG. 20B) showed that protein amounts in each group were nearly identical. (The blot used in FIG. 19B was stripped and sequentially reprobed with antibodies to Myc and GAPDH and so the lanes in FIG. 20 A and 20B are identical to FIG. 19B.

[000341] In another set of experiments, the stability of the canine IGVL3 -28-mouse C _K LC in transfected cells (FIG. 2 IB, reducing conditions) was examined in parallel with the secretion analysis (FIG. 21A, non-reducing conditions). Again, much less IgG2a was secreted when the LC was canine IGVL3 -28-mouse C _K (FIG. 2 A, 2102) than when it was canine IGVL3 -28-mouse Cxi (FIG. 2A, 2103) or IGVL3 -28-mouse Cx ₂ (FIG. 2A, 2104) However there was a significant amount of intracellular KLC in IGVL3-28-mouse C _K cell lysates detectable with an anti-k antibody (FIG. 2B, 2102) , similar to the levels seen when the LC was canine IGVK2-5-mouse C _K (FIG. 20B, 2105). Thus the hybrid IGVL3-28- mouse C _K was expressed well and not rapidly degraded intracellularly. In this particular canine VH-VK combination, the secretion of canine IgG2a using VK2-5 was similar when it was attached to V _K (2105), Cxi (2106) or Cx2 (2107).

[000342] The results in FIGs. 21A and 21B, indicate that the reduced secretion of Ig molecules bearing a hybrid canine Vx-mouse C _K was due to an inability to fold or to pair correctly with the y2a HC. While not wishing to be bound by theory, it is believed that this results in retention of the incompletely assembled IgG2a molecule in the endoplasmic reticulum (ER) by ER quality control mechanisms such as the Ig HC retention molecule BiP (Haas and Wabl (1983) Immunoglobulin Heavy Chain Binding Protein. Nature 306:387-389 PMID 6417546; Bole, et al. (1986) Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J. Cell Biology 102: 1558-1566 PMID 3084497).

Example 10: Expression of Partly Canine Immunoglobulin with Mouse IgD

[000343] IgD is co-expressed with IgM on mature B cells in most mammals. However, the issue of whether dogs have a functional constant region gene to encode the 6HC is quite controversial. Early serological studies using a mAb identified an“IgD-like” molecule that was expressed on canine lymphocytes (Yang, et al. (1995) Identification of a dog IgD-like molecule by a monoclonal antibody. Vet. Immunol and Immunopath. 47:215-224. PMID: 8571542). However, serum levels of this IgD increased upon immunization of dogs with ragweed extract. This is not typical of bona fide IgD, which is present in vanishingly small amounts in serum and is not boosted by immunization; IgD is primarily a BCR isotype, especially in mice. Later, Rogers, et al. ((2006) Molecular characterization of immunoglobulin D in mammals: immunoglobulin heavy constant delta genes in dogs, chimpanzees and four old world monkey species. Immunol. 118:88-100 (doi : 10.1111/j.1365-2567.2006.02345.x)) cloned a cDNA by RT-PCR of RNA isolated from dog blood that, by sequence homology, encoded an authentic 6HC. However, the most recent annotation of the canine IgH locus by the international ImMunoGeneTics information system® / www.iragt.org, (IMGT) lists C6 as a non-functional open reading frame because of a non-canonical splice donor site, NGC instead of NGT, for the hinge 2 exon. It is possible that some low level of correct“leaky” splicing and IgD expression may occur in the dog, thus accounting for the ability of Rogers, et al. to isolate a C6 cDNA clone. However, the concern was that the canine VH domains might not fold properly when linked to mouse C6, since the dog VH gene region has apparently been evolving with a partial or completely non-functional Cs gene. A problem with partial or absent assembly of the partly canine IgD could disturb normal B cell development. [000344] To test whether canine VH domains with a C6 backbone can assemble into an IgD molecule expressible on the cell membrane, transient transfection and flow cytometry analyses were conducting using methods similar to those described in Example 8.

[000345] 293T/17 cells were co-transfected with a human CD4 (hCD4) expression vector as a transfection control plus one of the HC constructs from Example 8, except that Cp was replaced with C6, and one of the k or l LC constructs, along with a CD79a/b expression vector. As can be seen in FIGS. 22-24, the HC with canine VH domains with a mouse IgD backbone were expressed on the cell surface when paired with a canine V _K -mouse C _K or a canine Cx-mouse Cx LC.

[000346] FIG. 22 shows expression of cell surface canine IGHV3-5 with a mouse IgD backbone and canine IGKV2-5/IGKJ1-C _K (column 2201) and canine IGLV3-28/IGLJ6 attached to mouse C i (2202), Cx2 (2203) or Cx ₃ (2204). In these studies, the top row (2205) shows staining for cell surface hCD4, the control for transfection efficiency. Row 2206 shows staining for CD79b, an obligate component of the BCR, which confirms cell surface IgD expression. Row 2207 shows IgD staining, 2208 shows k LC, and 2209 shows l LC. These particular canine VH/V _K or VH/VX LC combinations were expressed well on the cell surface.

[000347] FIG. 23 shows expression of cell surface canine IGHV3-19 with a mouse IgD backbone and canine IGKV2-5/IGKJ1-C _K (column 2301) and canine IGLV3-28/IGLJ6 attached to mouse Cxi (2302), Cx2 (2303) or Cx3 (2304). (The cell surface staining data is arranged the same as in FIG. 22.) The cell surface expression of IgD with these particular canine VH/V _K or VH/VX LC combinations was not as high as in FIG. 22. Recall that canine IGHV3-19 was also the most stringent VH in terms of its ability to associate with a canine V _K -mouse Cx LC. (FIG. 19).

[000348] FIG. 24 shows expression of cell surface canine IGHV4-1 with a mouse IgD backbone and canine IGKV2-5/IGKJ1-C _K (column 2401) and canine IGLV3-28/IGLJ6 attached to mouse Cxi (2402), Cx2 (2403) or Cx ₃ (2404). (The cell surface staining data is arranged the same as in FIG. 22.) The cell surface expression of IgD with these particular canine VH/V _K or VH/VX LC combinations was intermediate between that observed in FIG. 22 and FIG. 23. [000349] This data demonstrates that canine VH genes were expressed with a mouse IgD backbone, although the level of cell surface expression varied depending on the particular HC/LC combination. It is believed that HC/LC combinations that can be expressed as IgD on the cell surface are selected into the follicular B cell compartment during B cell development, generating an adequate BCR repertoire.

[000350] The preceding merely illustrates the principles of the methods described herein. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims that follow, unless the term "means" is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. §112 ]}6. All references cited herein are incorporated by reference in their entirety for all purposes.

SEQUENCE TABLES

Canine Ig

(NB, the sequence and annotation of the dog genome is still incomplete. These tables do not necessarily describe the complete canine VH, DH and JH, VK AND JK, or nl and JV gene segment repertoire.)

(F = Functional, ORF = open reading frame, P = pseudogene, *0X indicates the IMGT allele number)

Table 1. Canine IGH locus

Germline VH sequences

SEQ ID NO. 1 1 GH VI -4-1 (P)

>IGHVl-4-l* 01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtccagctggtgcagtctggggctgaggtgaggaaaccagtttcatctgtgaag gtc

tcctggaaggcatctggatacacctacatggatgcttatatgcactggttatgacaa gct

tcaggaataaggtttgggtgtatgggatggattggtcccaaagatggtgccacaaga tat

tcacagaagttccacagcagagtctccctgatggcagacatgtccaaagcacagcct aca

tgctgctgagcagtcagaggcctgaggacacacctgcatattactgtgtgggacact

SEQ ID NO. 2 IGHV1-15 (P)

>IGHV1-15*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtccagctggtgcagtctggggctgaggtgaagaagccaggtacatccgtgaag gtc

tcatgcaagacatctggatacaccttcactgactactatatgtactgggtacgacag gct

tcaggagcagggcttgattggatgggacagattggtccctaagatggtgccacaagg tat

gcacagaagtttcagggcagagtcaccctgtcaacagacacatccacaagcacagcc tac

atggagctgagcagtctgagagctgaggacacagccatgtactactctgtgaga

SEQ ID NO. 3 IGHV1-17 (P)

>IGHV1-17*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtccagctggtgcagtctggggctgaggtgaagaagctaagggcatcagtgata gtc

ccctgcaagacatctggatacagcttcactgactacattttggaatgggtatgacag gct

ccaggaccagggcttgagtggatgggatggattggtcctgaagatggtgagacaaag tat

gtgcagaagttccaggcagagtcaccctgatggcagacacaaccacaagcacagcca aca

tggagctgaccagtctgagagctgaggacacagccatgtactactgtgtga

SEQ ID NO. 4 IGHV1-30 (F)

>IGHV1-30*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtccagctggtgcagtctggggctgaggtgaagaagccaggggcatctgtgaag gtc

tcctgcaagacatctggatacaccttcattaactactatatgatctgggtacgacag gct

ccaggagcagggcttgattggatgggacagattgatcctgaagatggtgccacaagt tat

gcacagaagttccagggcagagtcaccctgacagcagacacatccacaagcacagcc tac

atggagctgagcagtctgagagctggggacatagctgtgtactactgtgcgaga

SEQ ID NO. 5 IGHV3-2 (F)

>IGHV3-2* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgaga etc

tcctgtgtggcctctggattcaccttcagtagcaactacatgagctggatccgccag gct

ccagggaaggggctgcagtgggtctcacaaattagcagtgatggaagtagcacaagc tac

gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctg tat

ctgcagatgaacagcctgagagatgaggacacggcagtgtattactgtgcaaggga SEQ ID NO. 6 IGHV3-3 (F)

>IGHV3-3* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagacatggtgaagcctggggggtccctgaga etc tcctgtgtggcctctggatttaccttcagtagttactacatgtattgggcccgccaggct ccagggaaggggcttcagtgggtctcacacattaacaaagatggaagtagcacaagctat gcagacgctgtgaagggccgattcaccatctccagagacaacgcaaagaatacgctgtat ctgcagatgaacagcctgagagctgaggacacagcggtgtattactgtgcaaagga

SEQ ID NO. 7 IGHV3-4 (P)

>IGHV3-4* 01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtgcagctggtggagtctgggggagacctgatgaagcctgggggggtccctgag act ctcctgtgtggcctctgaattcatcttcagtggctactggaagtactggatccaccaagc tccagggaaggggctgcagtgggtcacatggattagcaatgatggaagtagcaaaagcta tgcagacgctgtgaagggccaattcaccatctccaaagacaatgccaaatacacgctgta tctgcagatgaacagcctgagagccgaggacatggccgtgtattactgtatgatgca

SEQ ID NO. 8 IGHV3-5 (F)

>IMGT000001 I IGHV3-5*01 I Canis lupus familiarisjooxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgagactt tcctgtgtggcctctggattcaccttcagtagctaccacatgagctgggtccgccaggct ccagggaaggggcttcagtgggtcgcatacattaacagtggtggaagtagcacaagctat gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtat cttcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagtga

SEQ ID NO. 9 IGHV3-5-1 (P)

>IMGT000001 I IGHV3-5-l*01 | Canis lupus familiarisjooxer | P | V-REGION | gaggtgcagctggtggagtctgggggagccctggtgaagcctgggggggtccctgagact ctcctatgtggcctctggattcaccttcagtagctaccacatgagctgggtccgccaggc tccagggaaggggctgcagtgggtcgcatacattaacagtggtggaagtagggatccctg ggtggcgcagtggtttggcgcctgcctttggcccagggcacgatcctggagacccgggat cgaatcccacgtcgggctccctgcatggagcctgcttctccctctgcctgtgtctct

SEQ ID NO. 10 IGHV3-6 (F)

>IGHV3-6* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgaga etc tcctgtgtagcctctggattcaccttcagtagctccgacatgagctggatccgccaggct ccaggaaaggggcttcagtgggtcgcatacattagcaatgatggaagtagcacaagctac gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctctat ctgcagatgaacagcctcagagccgaggacacggccgtgtattactgtgcaga

SEQ ID NO. 11 IGHV3-7 (F)

>IGHV3-7* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggagcaactggtggagtttggaggacacatggtgaatcctgggggttccctgggt ctc tcctgtcaggcctctggattcaccttcagtagctatggcatgagctgggtccgccaggct caaaagaaggggctgcagtgggtcggacatattagctatgatggaagtagtacatactac gcagacactttgagggacagattcaccatctccagagacaacaccaagaacatgctgtat ctgcagatgaacagcctgagagccgaggacacagccgtgtattactgcatgaggaa

SEQ ID NO. 12 IGHV3-8 (F)

>IGHV3- 8* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgaga etc tcctgtgtggcctctggattcaccttcagtaactacgaaatgtactgggtccgccaggct ccagggaaagggctggagtgggtcgcaaggatttatgagagtggaagtaccacatactat gcagaagctgtaaagggccgattcaccatctccagagacaacgccaagaacatggcgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagtga

SEQ ID NO. 13 IGHV3-9 (F)

>IGHV3- 9* 01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctggaggagacctggtgaagcctggggggtccctgaga ctt tcctgtgtggcctctggattcaccttcagtagctatgacatggactgggtccgccaggct ccagggaaggggctgcagtggctctcagaaattagcagtagtggaagtagcacatactac gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcaaggga

SEQ ID NO. 14 IGHV3-10 (F)

>IGHV3-10*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagactgagggagacctggtgaagcctgggggatccctgaga ctt tcctgtgtggcctctggattcaccttcagtagctacgacatggactgggtctaccaggct ccagggaaagggttacagtgggtcacatacattagcaatggtggaagtagcacaaggtat gcagacgctgtgaagggccaattcaccatctccagagacaacgccaggaacacgctctat ctgcagatgaacagcctgagagacaaggacatggccgtgtattactgtgtgagtga

SEQ ID NO. 15 IGHV3-11 (P)

>IMGT000001 I IGHV3-11* 01 | Canis lupus familiarisjooxer | P | V-REGION | gaggtgcagctggtggagtctaggggagacgtggtgaagcctggggaggtccctctcctg tgtggcctctagattcaccttcagtagctactacatgggctgggtccactaggctccagg gaaggggctgcagtgggtcgcaggtattaccaatgatagaagtagcacaagctatgcaga cgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtatctgca gatgaacagcctgggagccgaggacacggctgtgtattattgtgtgaaacaga

SEQ ID NO. 16 IGHV3-12 (P)

>IGHV3-12*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtgcagctggtggagtctggggagacctggtgaagcctggggggtctctgagac tct cctgtgtggcctctggattcaccttcagtagctactacatgagctgggtccgccaggctc cagggaaggggctgcagtgggtcggatacattaacagtggtggaagtagcacatactatg cagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtatc tgcagatgaacagcctgagagccgaggacacagctgtgtattactgtgggaaggga

SEQ ID NO. 17 IGHV3-13 (F)

>IGHV3-13*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggagcaactggtggagtttggaggacacatggtgaatcctgggggttccctgggt ctc tcctgtcaggcctctggattcaccttcagtagctatggcatgagctgggtccgccaggct caaaagaaggggctgcagtgggtcggacatattagctatgatggaagtagcacatactac acagacactgtgagggacagattcaccatctccagagacaacaccaagaacatgctgtat ctgcagatgaacagcctgagagccgaggacacagccgtgtattactgcatgaggaa

SEQ ID NO. 18 IGHV3-14 (P)

>IGHV3-14*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtgcagatggtggagtctgggggagacctggtgaagcctgggggatccctgaga etc tcctgtgtggcctctggattcaccttcagtaactacaaaatgtactgggtccaccaggct ccagggaaagggctggagtgggtcgcaaggatttatgagagtggaagtaccacatactac gcagaagctgtaaagggccgattcaccatctccagagacaacgccaagaacatggtgtat ctgcagatgaacagcctgagagcctaggacacggccgtgtattactgtgtgagtga

SEQ ID NO. 19 IGHV3-16 (F)

>IGHV3-16*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtacagctggtggagtctggaggagacctggtgaagcctggggggtccctgaga ctc tcctgtgtggcctctggattcacctttagtagttactacatgttttggatccgccaggca ccagggaagggcaatcagtgggtcggatatattaacaaagatggaagtagcacatactac ccagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacactgtat ctgcagatgaacagcctgacagtggaggacacagccctttattactgtgcgagaga

SEQ ID NO. 20 IGHV3-18 (F)

>IGHV3-18*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagaccttgtgaaacctgaggggtccctgaga etc tcctgtgtggtctctggcttcaccttcagtagctacgacatgagctgggtccgccaggct ccagggaaggggctgcagtgggtcgcatacattagcagtgatggaaggagcacaagttac acagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagaactgaggacacagccgtgtattactgtgcgaagga SEQ ID NO. 21 IGHV3-19 (F)

>IGHV3-19*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctgcggggtccctgagactg tcctgtgtggcctctggattcaccttcagtagctacagcatgagctgggtccgccaggct cctgagaaggggctgcagttggtcgcaggtattaacagcggtggaagtagcacatactac acagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacagtgtat ctgcagatgaacagcctgagagccgaggacacggccatgtattactgtgcaaagga

SEQ ID NO. 22 IGHV3-20 (P)

>IGHV3-20*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctgggggatacctggtgaagcctggagggtcctgagactct cctctgtgtcctctggattcaccttcagtatctactgcatgtgatgggtctgccaggctc caggaaaggggctgcagtgagtcgcatacagtaacagtggtggaagtagcactaggtaca cagacgctgtgaagggctgattcaccacctccagagacaatgccaagaacacactgtatc tgcagatgaacagcctgagagtgaggacacagcggtgtattactgtgcaggtga

SEQ ID NO. 23 IGHV3-21 (P)

>IGHV3-21*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctgttggagtctgggggagacctggtgaagcctggggggtccctgagactg tcctgtgtggtctctggattcaccttcagtaagtatggcatgagctgggtctgccaggct ttggggaaggggctacagttggtcgcagctattagctaagatggaaggagcacatactac acagacactgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtac ctgcagatgaacagcttgagagctgaggacacggccgtgtattactgtgagagtga

SEQ ID NO. 24 IGHV3-21-1 (P)

>IGHV3-21-1*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgaagctagtggagtctgggggagacctggtgaagcctgggggatcaattagactc tcctatgtgacctctggattcaccttcaggagctactggatgagctgggtcagccaggct ccagggaaggggctgcagtgggtcatatgggttaatactggtggaagcagaaaaagctat gcagatgctgtgaaggggtgattcaccatctccagagacaatgccaagaacacgctgtat ctgcatatgaacagcctgagagccctgtattattatgtgagtga

SEQ ID NO. 25 IGHV3-22 (P)

>IGHV3-22*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagatgatggagtctgggggagaactgatgaagcctgcaggatccctgagacct cctgtgtggcctctggattcaccttcagtagctactggatgtactggatccaccaaactc cggggaaggggctgcagtgggtcgcaggtattagcacagatggaagtagcacaagctacg tagacgctctgaagggctgattcaccatctccagagacaacgccaagaacacgctctatc tgcagatgaacagcctgagagccgaggacatggccatgtattactgtgcaga

SEQ ID NO. 26 IGHV3-23 (F)

>IGHV3-23*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggagaagcctgggggatccctgagactg tcctgtgtggcctctggattcaccttcagtagctacggcatgagctgggtccgccaggct ccagggaaggggctgcagggggtctcattgattaggtatgatggaagtagcacaaggtat gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacagccgtgtattcctgtgcgaagga

SEQ ID NO. 27 IGHV3-24 (F)

>IGHV3-24*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagaccttgtgaagcctgaggggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagcttctacatgagctggttctgccaggct ccaaggaaggggctacagtgggttgcagaaattagcagtagtggaagtagcacaagctac gcagacattgtgaagggccgattcaccatctccagagacaatgccaagaacatgctgtat ctgcagatgaacagcctgagagccgaggacatggccgtatattattgtgcaaggta SEQ ID NO. 28 IGHV3-25 (P)

>IGHV3-25*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagcctgggggagaactggtgaagcctggggcgtccctgagaetc tcctgtgtggtccctggattcaccttcagtagctacaacatgggctgggctcaccagcct ccagggaaggggatgcagtgggtcgcaggttttaacagcggtggaagtagcacaagctac acagatgctgtgaagggtgaattcaccatctccagagacaatgtcaagaacacgctgtat ctgcagatgaacagcctgagatccgaggacacggccgtgtattactgtgtgaagga

SEQ ID NO. 29 IGHV3-26 (P)

>IGHV3-26*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgtagctggtggagtctgggggagacctggtgaagcctggggggtccctgagaetc tcctgtgtgggctctggattcaccttcagtagctactggatgagctgggtccgccaggct ccagggaaggggctacagtgggttgcagaaattagcggtagtggaagtagcacaaactat gcagacgctgtgaagggccgattcatcatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccatgtattactgtgcaaggga

SEQ ID NO. 30 IGHV3-27 (P)

>IGHV3-27*01 I Canis lupus familiaris boxer | P | V-REGION | aaggtgcatctggtggagtctgcgggagacgtggtgaagcctaggaggtccctgagactc tcctgtgtgggctctggattcaccttcagtagctacagcatgtggtgggcccgtgaggct cccgggatggggctacagggggtcgcaggtattagatatgatggaagtagcacaagctac gcagacgctctgaagggccgattcaccatctccagagacaatgccaaaaacacactgtat ctgtagaagaacagcctgagagccgagggaggacacggccgtgtattactgtgcgaggga

SEQ ID NO. 31 IGHV3-28 (P)

>IGHV3-28*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctagtggagtctgggggagacctggtgaagtctgggggggtccctgagagt ctcctgtgtgggctctggattcaccttcagtagctactggatgtactgggtccaccaggc tccagggaaggggctccatgggtcgcatggattaggtatgatggaagtagcacaagctac gcagaagctgtgaaaggccgattcactgtttctagagacaacgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgtgaggga

SEQ ID NO. 32 IGHV3-29 (P)

>IGHV3-29*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtcctggggagacctggtgaagactggaggtttcctgagaetc tcctgtctcctgtgtggcttccggattcaccttcagtaactacagcatgatctgggtccg ccaggctccaaggaaggggctgcagtggatcacaactattagcaatagtggaagtagcac aaatcacgcagacacagtaaagggccgatttaccatctccagagacaacaccaagaacac gctgtatctacagatgagcagcctgggagccgatgacacggccctgtattactgtgtgag gga

SEQ ID NO. 33 IGHV3-31 (P)

>IGHV3-31*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctgggggagaactggtgaagcctggggggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagctactacatgagctggatccgccaggct cctgggaaggggctgcagtgggtcgcagatattagtgacagtggaggtagcacatactac actgacgctgtgaagggccgattcaccatctccagagacaacgtcaagaactcgctgtat ttgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaagga

SEQ ID NO. 34 IGHV3-32 (ORF)

>IGHV3-32*01 I Canis lupus familiaris boxer | ORF | V-REGION | ggggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgacactc tcctgtgtggcctatggattcaccttcagtagctacagcatgcaatgggtctgtcaggct ccagggaagggggtgcagtgggtcgcatacattaacagtggtggaagtagcacaagctcc gcagatgctgtgaagggtcgattcatcatctccagagacaacgtcaagaacacgctatat ctgcagatgaacagcctgagagccgaggacaccgccgtgtattactgtgcgggtga

SEQ ID NO. 35 IGHV3-33 (P)

>IGHV3-33*01 I Canis lupus familiaris boxer | P | V-REGION | gagatgcagctggtggaggctgggggagacctggtgaagcttggggggtccctgagaetc ttctgtgtggcctctggatttaccttcagtagctattggatgagctgggtcggccaggct ccagggaaagggttgcagtgggttgcatacattaacagtggtggaagtagcacatactat gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaactgcctgagagccgaggacacggccgtatattactgtgtggga

SEQ ID NO. 36 IGHV3-34 (F)

>IGHV3-34*01 I Canis lupus familiaris boxer | F | V-REGION | cagacactgtgaagggccgattcaccatctccagagacaacgccaagaacacgctctatc tgcagatgaacagcctgagagctgaggacacggccgtgtattactgtgcgaagga (Incomplete sequence in database)

SEQ ID NO. 37 IGHV3-35 (F)

>IGHV3-35*01 I Canis lupus familiaris boxer | F | V-REGION | | | gaggtgcagctggtggagtctgggggagacctggtgaagcctgtgggatccctgagaetc tcctgtgtggcctctggattcaccttcagtagctatgacatgaactgggtccgccaggct ccagggaaggggctgcagtgggtcgcatacattagcagtggtggaagtagcacatactat gcagatgctgtgaagggccggttcaccatctccagagacaacgccaagaacacgctgtat cttcagatgaacagcctgagagccgaggacacggccatgtattactgtgcgggtga

SEQ ID NO. 38 IGHV3-36 (P)

>IGHV3-36*01 I Canis lupus familiaris boxer | P | V-REGION | gaggggcagctggcggagtctgggggagacctggtgaagcctgagaggtccctgagaetc gcccgtgtggcctctggattcaccttcatttcctataccatgagctgggtccacaaggct cctgggaaggggctgccgtgagtcgcatgaatttattctagtggaagtaacatgagctat gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacatgctgtat ctgcagatgaacagcctgagagctgaggacatggccatgtattactgtgtgaatga

SEQ ID NO. 39 IGHV3-37 (F)

>IGHV3-37*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtacagctggtggagtctggggaagatttggtgaagcctggagggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagcagtgaaatgagctgggtccaccaggct ccagggcaggggctgcagtgggtctcatggattaggtatgatggaagtatctcaaggtat gcagacactgtgaagggccgattcaccatctccagagacaatgtcaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccatatattactgtgcaga

SEQ ID NO. 40 IGHV3-38 (F)

>IGHV3-38*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggaccttgagactg tcctgtgtggcctctggattcacctttagtagctatgacatgagctgggtccgtcagtct ccagggaaggggctgcagtgggtcgcagttatttggaatgatggaagtagcacatactac gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaagga

SEQ ID NO. 41 IGHV3-39 (F)

>IGHV3-39*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtacagctggtggaatctgggggagacctcgtgaagcctgggggttccctgagactc tcctgtgtggcctcgggattcaccttcagtagctactacatgagctggatccgccaggct cctgggaaggggctgcagtgggtcgcagatattagtgatagtggaggtagcacaggctac gcagacgctgtgaagggccggttcaccatctccagagagaacgccaagaacaagctgtat cttcagatgaacagcctgagagccgaggacacagccgtgtattactgtgcgaagga

SEQ ID NO. 42 IGHV3-40 (P)

>IGHV3-40*01 I Canis lupus familiaris boxer | P | V-REGION | atgcaatgggtccgtcaggctcctgggaagggggtgcagtgggtcgcatacattaacagt ggtggaagtagcacaagcttcgcagatgctgtgaagggcatgagctggtttcgccaggct ccagggaaggggctgcaatgggttacatggattgggtatgatggaagtagcacatactac acagacactgtaaagggccgattcactatctccatagacaacgccaagaacatgctgtat ctgcagatgaacagcctgagagccgaggacatagccctgtattactgtgcgaggga SEQ ID NO. 43 IGHV3-41 (F)

>IGHV3-41*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgagaetc tcctgtgtagcctctggattcaccttcagtaactacgacatgagctgggtccgccaggct cctgggaaggggctgcagtgggtcgcagctattagctatgatggaagtagcacatactac actgacgctgtgaagggccgattcaccatctccagagacaacgccaggaacacagtgtat ctgcagatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaagga

SEQ ID NO. 44 IGHV3-42 (P)

IGHV3-42* 01 I Canis lupus familiaris boxer | P | V-REGION | gaagtgcagctggtggagtctgggggaagacctggtgaagccaggggggtccctgagact ctcctgtgtgacctctggattcaccttcagtaggtatgccatgagctgggtcggccaggc tccagggaagggcctgcagtgggttgcagctattagcagtagtggaagtagcacatacta cgtagatgctgtgaagggccgattcaccatctccatagacaacgccaagaacatggtgta tctgcagatgaacagcctgagagctgaggatattgctgtgtattactgtgggaagga

SEQ ID NO. 45 IGHV3-43 (P)

>IGHV3-43*01 I Canis lupus familiaris boxer | P | V-REGION | aaggtgtagctggtggagtctgggggagacctgatgaagcctgggggttccctgagactg tcctgtgtggcctctggattcaccttcaggagctatggcatgagctgggtctgccaggct tcagggaaggggctgcagtgggtcgcagctattagctatgatggaaggagcacatactac acagacactgtgaagggccgattcaccatctccagagacaatggcaagaacacgctgtac ctgcagatgaacagcttgagagctgaggacacggccgtgtattactgtgcgagtga

SEQ ID NO. 46 IGHV3-44 (ORF)

>IGHV3-44*01 I Canis lupus familiaris boxer | ORF | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctgggggttccctgagaetc tcatgtgtgacttctggattcaccttcagtagctattggatgagctgtgtccgccaggct ccagggaaggagctgcagtgggtcgcgtacattaacagtggtggaagtagcacatggtac acagacgctgtgaagggtcgattcaccatctccagagacaacgccaagaacacgctgtat ctgcagatgaacaacctgagagccgaagacacggccgtgtattactgtgcgaggga

SEQ ID NO. 47 IGHV3-45 (P)

>IGHV3-45*01 I Canis lupus familiaris boxer | P | V-REGION | gaagtacagctgctggagtctgggggagaccgagtgaaacctggggggtcccagagactc tcctgtgtggcctcaaggttcaccttcagtagctacagcatgcattgtctccgtcagtct cctgggatggggctacagtgggtcacatacattagcagtaatggaagcagcacatactat gcagacgctgtgaagggtcgattcaccatctccagagacaaagccaagaacatgctttat ctacagatgaacagcctgagagctcaggacatagccctgtattactgtgcagatg

SEQ ID NO. 48 IGHV3-46 (F)

>IGHV3-46*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtacagctggtggagtctggggaagatttggtgaagcctggagggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagcagtgaaatgagctgggtccaccaggct ccagggcaggggctgcagtgggtctcatggattaggtatgatggaagtagctcaaggtat gcagacactgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccatatattactgtgcaga

SEQ ID NO. 49 IGHV3-47 (F)

>IGHV3-47*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggcgaagcctggggggtccctgagaetc tcctgtgtggcctctggattaaccttcagtagctacagcatgagctgggtccgccaggct cctgggaaggggctgcagtgggtcacagctattagctatgatggaagtagcacatactac actgacgctgtgaagggccgattcaccatctccagagacaacgccaggaacacagtgtat ctgcagatgaacagcctgagagccgaggacacagctgtgtattactgtgtgga

SEQ ID NO. 50 IGHV3-47-1 (P)

>IGHV3-47-l*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgccactggtggaatctgggggagagctggtgaagcctggggggtccctgagactc tcctttgtagcctctgcattcactttcagtagttactggataagctgggtccgccaagct ccagggaaagggctgcactgagtctcagtaattaacaaagatggaagtaccacataccac gcagatgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagctgaggacacggctgtgtattactgtgcaca

SEQ ID NO. 51 IGHV3-48 (P)

>IGHV3-48*01 I Canis lupus familiaris boxer | P | V-REGION | gaggagcagttggtgaaatctaggggagacctggtgaagcctggcgggtccctgagaetc ttctgtgagtcctctacattcacctttcatagcaacagcatacattggctccaccagtct cccggtagtggctacagtgggtcatatccaatagcagtaatggaagtagcatgtactatg cagacgctgtaaagggctgattcaccatctccagagacagcaccaggaacatgctgtatc tgcagatgaacagcctgagagctgaggacacagccgtgcattgctgtgcgaggga

SEQ ID NO. 52 IGHV3-49 (P)

>IGHV3-49*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctgggggagacctcatgaagcctggggggtccctgagaetc tcctgtgtggccgctggattcaccttcagtagctacagcatgagctgggtccgccaggct cccgggaaggggattcagtgggtcgcatggatttaagctagtggaaatagcacaagctac acagatgctgtgaagggccgattcaccatctccagagaacgccaagaacacagtgtttct gcagatgaacagcctgagagctgaggacaaggccatgtattactgtgcgaggga

SEQ ID NO. 53 IGHV3-50 (F)

>IGHV3-50*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccttgagaetc tcctgtgtggcctctggtttcaccttcagtagcaacgacatggactgggtccgccaggct ccagggaaggggctgcagtggctcacacggattagcaatgatggaaggagcacaggctac gcagatgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtat ctgcagatgaacagcctgagagctgaggacacagccgtgtattactgtgcgaagga

SEQ ID NO. 54 IGHV3-51 (P)

>IGHV3-51*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggaggagtctgggggagacctggtgaagcctggggttccctaagactgt cctgtgtgacctccggattcactttcagtagctatgccatgcactgggtccgccaggctc cagggaaggggctgcagtgggtcgcagttattagcagggatggaagtagcacaaactacg cagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacatgctgtatc tacagatgaacagcctgagagctgaggacacggccatgtattactgtgcgaagga

SEQ ID NO. 55 IGHV3-52 (P)

>IGHV3-52*01 I Canis lupus familiaris boxer | P | V-REGION | | gaagtgcagctggtggagtatgggggagagctggtgaagcctggggggtccctgagactg tcctgtgtggcctccggattcaccttcagtatctactacatgcactgggtccaccaggct ccagggaaggggctgcagtggttcgcatgaattaggagtgatggaagtagcacatactac actgatgctgtgaagggccgattcaccatctccagagacaattccaagaacactctgtat ctgcagatgaccagcctgagagccgaggacacggccctatattactgtgcgatgga

SEQ ID NO. 56 IGHV3-53 (P)

>IGHV3-53*01 I Canis lupus familiaris boxer | P | V-REGION | gagatgcagctggtggagtctagggaggcctggtgaagcctggggggtccctgagactct cctgtgtggaccctggattcaccttcagtagctactggatgtactgggtccaccaggctc cagggatggggctgcagtggcttgcagaaattagcagtactggaagtagcacaaactatg cagacgctgtgaggggcccattcaccatctccagagacaatgccaagaacacgctgtacc tgcaggtgaacagcctgagagccgaagacacggccgtgtattactgtgtgagtga

SEQ ID NO. 57 IGHV3-54 (F)

>IGHV3-54*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctgatgaagcctggggggtccctgagaetc tcctgtgtggcctccggattcactatcagtagcaactacatgaactgggtccgccaggct ccagggaaggggctgcagtgggtcggatacattagcagtgatggaagtagcacaagctat gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgtgaaggga SEQ ID NO. 58 IGHV3-55 (P)

>IGHV3-55*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctggggaaacctggtgaagcctggggagtctctgagactct cttgtgtggcctctggattcaccttcagtagctactggatgcattgggtctgccaggctc cagggaaagggttggggtgggttgcaattattaacagtggtggaggtagcacatactatg cagacacagtgaagggccaattcaccatcttcagagacaatgccaagaacatgctgtatc tgcagatgaacagcctgagagcccaggacatgaccgcgtattactgtgtgagtga

SEQ ID NO. 59 IGHV3-56 (P)

>IGHV3-56*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggaatctgggggagacctggtgaagcctgggggatccctgagactc tcctgtgtggcctctggattcaccttcagtagctactatatggaatgggtctgccaggct ccagggaggggctgaagtgggtcgcacggattagcagtgacggaagtagcacatactaca cagacgctgtgaagggccgattcaccatctccagagacaatgccaagacggccgtgtatt actgtgcgaagga

SEQ ID NO. 60 IGHV3-57 (P)

>IGHV3-57*01 I Canis lupus familiaris boxer | P | V-REGION | gaagtgcagcttgtggagtctgggggagagctggtgaagcctgggggttccctgagactg tcctgtgtggcctctggattcaccttcagtagctactacatgcactgggtctgcaggctc cagggaaggggctgcagtgggttgcaagaattaggagtgatggaagtagcacaagctacc cagacgctgtgaagggcagattcaccatctccagagacaattccaagaacactctgtatc tgcagatgaacagcctgagagctgatgatacggccctatattactgtgcaaggga

SEQ ID NO. 61 IGHV3-58 (F)

>IGHV3-58*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagacctggtgaagcctgggggatccctgagaetc tcttgtgtggcctccggattcaccttcagtagccatgccaagagctgggtccgccaggct ccagggaaggggctgaagtgggtagcagttattagcagtagtggaagtagcacaggctcc gcagacactgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagctgaggacacagccgtgtattactgtgcgaagga

SEQ ID NO. 62 IGHV3-59 (P)

>IGHV3-59*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtacagctggtggagtctggaggagaccttgtgaagactgagcggtccctgagactc tcctgtgtggcctctggattcaccttcagtagcttctacatgaggtgtctgccagactcc agggaagggactacagtgggttgcagaaattagcagtagtggaagtagcacaagctacac agatgctctgaagggctgattctccatctccaaaaacaatgccaagaacacgctgtatct gcagatgaacagcctgagagccgaggtcacagccgtatattactgtgcaaggta

SEQ ID NO. 63 IGHV3-60 (P)

>IGHV3-60*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgaagctggtggagtctgggggagacctgttgaagcctgggggatcaattaaactc tcctatgtgacctctggattcaccttcaggagctactggatgagctgggtcagccaggct ccagggaaggggctgcagtgggtcacatgggttaatactggtggaagcagcaaaagctat gcagatgctgtgaaggggcaattcaccatctccagagacaatgccaagaacacgctgtat ctgcatatgaacagcctgatagccctgtattattgtgtgagtga

SEQ ID NO. 64 IGHV3-61 (F)

>IGHV3-61*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctggtggaaacctggtgaagcctgggggttccctgagactg tcctgtgtggcctctggattaaccttctatagctatgccatttactgggtccacgaggct cctgggaaggggctgcagtgggtcgcagctattaccactgatggaagtagcacatactac actgacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagctgaggacatgcccgtgtattactgtgcgaggga

SEQ ID NO. 65 IGHV3-62 (P)

>IGHV3-62*01 I Canis lupus familiaris boxer | P | V-REGION | gaggagcagctggtggagtctcggggagatctggtgaagtctggggggtccctgagaetc tcctgtgtggccccttgattcaccttcagtaactgtgacatgagctgggtccattaggct ccaggaaagggctgcagtgtgttgcatacattagctatgatggaagtagcacaggttaca aagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacatgctgtatc ttcagatgaacagcctgagagctgaggacacggctctgtattactgtgcaga

SEQ ID NO. 66 IGHV3-63 (P)

IGHV3-63* 01 I Canis lupus familiaris boxer | P | V-REGION |

gaggagcagttggtgaaatctaggggagacctggtgaagcctggcgggtccctgaga etc ttctgtgagtcctctacgttcacctttcatagctacagcatgcattggctccaccagtct cccggtagtggctacagtgggtcatatccaatagcagtaatggaagtagcatgtactatg cagacgctgtaaagggctgatacaccatctccagagacaacaccaggaacatgctgtatc tgcagatgaataacctgagagctgaggacacagccgtgcattgctgtgcgaggga

SEQ ID NO. 67 IGHV3-64 (P)

>IGHV3-64*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtgcagctggtggagtctgcgggagaccccgtgaagcctggggggtccctgaga etc tcctgtgtggccgctggattcaccttcagtagctacagcatgagctgggtccgccaggct cccgggaaggggatgcagtgggtcgcatggatatatgctagcggaagtagcacaagctac gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacactgttt ctgcagatgcctgagagctgaggacacggccatgtattcctgtgcagggga

SEQ ID NO. 68 IGHV3-65 (P)

>IGHV3-65*01 I Canis lupus familiaris boxer | P | V—

REGION Igatgtacagctggtggagtctgggggagacctggtgaagcctggggggtccctgagact g tcctgtgtggcctctggattcacctgcagtagctactacatgtactagacccaccaaatt ccagggaaggggatgcagggggttgcacggattagctatgatggaagtagcacaagctac accgacgcaatgaaaggccgattcaccatctccagagacaacgccaagaacatgctgtat ctgcaatgaacagcctgagagccgaggacacagccgtgtattactgtgtgaagga

SEQ ID NO. 69 IGHV3-66 (P)

>IGHV3-66*01 I Canis lupus familiaris boxer | P | V-REGION |

gaggtgcagctggtggagtctggcggagacctggtgaagcctgggcggtccctgaga ctg tcctgtatggcctctggattcacttcagtagctacagcatgagctgtgtccgccaggctc ctgggaagggctgcagtgggtcgcaaaaattagcaatagtggaagtagcacatactacac agatgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctctatct gcagatgaacagcctgagagccgaggacacggccttgtattactgtgcaga

SEQ ID NO. 70 IGHV3-67 (F)

>IGHV3-67*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtgcagctggtggagtctgggggagacctggtgaagcctggggggtccctgaga ctg tcctgtgtggcctctggattcaccttcagtagctactacatgtactgggtccgccaggct ccagggaaggggcttcagtgggtcgcacggattagcagtgatggaagtagcacatactac gcagacgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagccgaggacacggctatgtattactgtgcaaagga

SEQ ID NO. 71 IGHV3-68 (P)

>IGHV3-68*01 I Canis lupus familiaris boxer | P | V-REGION |

gaagtgcagctggtggagtctgggggagagctggtgaagcctggggggtccctgaga etc tcctgtgtggcctctggattcaccttcagtagctactacatgtactgggtccgccaggct ccagggaaatggctgctgtgggtcacatgaattaggagtgatggaagtagcacatataca ctgatgctgtgaaggaccgatacaccatctccaaagacaattccaagaacattctgtatc tgcagatgaacagcctgagagccaaggacacggccctatatccctgtgcaatgga

SEQ ID NO. 72 IGHV3-69 (F)

>IGHV3-69*01 I Canis lupus familiaris boxer | F | V-REGION |

gaggtacagctggtggagtctgggggagacctggtgaagcctgggggatccctgaga ctg tcctgtgtggcctctggattcaccttcagtagctatgccatgagctgggtccgccaggct ccagggaaggggctgcagtgggtcgcatacattaacagtggtggaagtagcacatactac gcagatgctgtgaagggccggttcaccatctccagagacaatgccaggaacacactgtat ctgcagatgaacagcctgagatccgaggacacagccgtgtattactgtccgaagga SEQ ID NO. 73 IGHV3-70 (F)

>IGHV3-70*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctggaggagaccttgtgaagcctgagcggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagcttctacatgagctggttctgccaggct ccagggaaggggctacagtgtgttgcagaaattagcagtagtggaaatagcacaagctac gcagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtat ctacggatgcacagcctgagagccgaggacacggctgtatattactgtgcaaggta

SEQ ID NO. 74 IGHV3-71 (P)

>IGHV3-71*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgaagctggtggagtgtgggggagacctggtgaagcccgggggatcgattagaetc tcctttgtgacctctggattcaccttcaggagctattggatgggctgtgtcagccaggct ccagggaaggggctgcagtgggtcacatgggttaatactggtggaagcagcaaaagctat gcagatgctatgaaggggcgatttaccatctccaggcacaaagccaagaacacactatct gcatatgaacagcctgagagccgtgtattattgtgtgagtga

SEQ ID NO. 75 IGHV3-72 (P)

>IGHV3-72*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctggcggagacctggtgaagcctggggattccctgagactg tcctgtgtggcctctggattcaccttcagtagctatgccatgagctgggtccgccaggct cctaggaaggggctgcagtgggtcggatacattagcagtgatggaagtagcacataatac gcagacgctgtgaagggccgattcaccatttccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagctgaggatacggccctgtataactgtgcaaggga

SEQ ID NO. 76 IGHV3-73 (P)

>IGHV3-73*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctgatggagtctgggggagacctggtgaagcctggggggtccctgagaetc tcctgtgtggcccctggattcaccttcagtaactatgacatgagctcggtccattagact ccaggaaagggctgcagtgtattgcatatattagctatgatggaagtagcacaggttaca aagacgctgtgcagggccgattcaccatctccagagacaacgccaagaacacgctgtatc ttcagatgaacagcctgagagctgagcacacggccctgtattactgtgcaga

SEQ ID NO. 77 IGHV3-74 (P)

>IGHV3-74*01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctgggggagacttggtgaagccttgtgggctcctgagaetc tcctgtgtggcttctggattcaccttcagtagctacatcatgagctgggtccgccaggct ccagggaagtggctgcagtgggtcgcatacattaacagtggtggaagtagcacaaggtac acagatgctgtgaagggccgattcacctctccagagacaacgccaagaacatgctgtatc tgcagttgaacagcctgagagccgaggacaccgctgtgtattactgtgcgaggga

SEQ ID NO. 78 IGHV3-75 (F)

>IGHV3-75*01 I Canis lupus familiaris boxer | F | V-REGION | gaattgcagctggtggagcttgggggagatctggtgaagccaggggggtccctgagaetc tcctgtgtggcctctggattcaccttcagtagctatgccatgagttgggtctgccaggct ccagggaaggggctgcagtgggttgcagctattagcagtagtggaagtagcacataccat gtagacgctgtgaagggccgattcaccatctccagagacaacgccaagaacacagtgtat ctgcagatgaacagcctgagagccgaggacacggccgtgtattactgtgcaga

SEQ ID NO. 79 IGHV3-76 (F)

>IGHV3-76*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgccactggtggaatctgggggagagctggtgaagcctgaggggtccctgagattc tcctgtgtagcctctggattcactttcagtagttactggataagctgggtccgccaagct ccagggaaagggctgcactgggtctcagtaattaacaaagatggaagtaccacataccac gcagatgctgtgaagggccgattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgagagctgagggcacgactgtgtattactgtgcaca

SEQ ID NO. 80 IGHV3-77 (P)

>IGHV3-77*01 I Canis lupus familiaris boxer | P | V-REGION | gaggagcagttggtgaagtctgggggagacctggtgaagcttggcaggtccctgagtcct ctacattcacctttcatagctacagcatgcattggctccaccagtctcccggtagtggct acagtgggtcatatccaatagcagtaatggaagtagcatgtactatgcagacgctgtaaa gggttgattcaccatctccagagacaacaccaggaacacgctgtatctgcagatgaacag cctgagagccgacgacacggccgtgtgttgctgtgcgaggga

SEQ ID NO. 81 IGHV3-78 (P)

> I IGHV3-78* 01 I Canis lupus familiaris boxer | P | V-REGION | gaggtgcagctggtggagtctgggggagaccttgtgaagccggaggggtccctgagaetc tcctgtgtggccgctggattcacctttagtagctacagcatgagctgggtccgccaggct cccgggaagggggtgcagtgggtcacatagatttatgctagtggaagtagcacaagctac acagatgctgtgaagggccgattcaccatctccagagacaacgccaagaacacagtgttt ctgcagatgaacagcctgagagctgagaacacggccatgtattcctgtgcaaggga

SEQ ID NO. 82 IGHV3-79 (P)

>IGHV3-79*01 I Canis lupus familiaris boxer | P | V-REGION | tggggaattccctctggtgtggcctctggattcacctgcagtagctccctcacctccctc tcctgtgtggcctctagattcaccttcagtagctactacatatactgtatccaccaagct ccagggaaggggctgcaggtggtcgcatggattagctatgatggaagtagaacaagctac gccgacgctatgtagggccaattcatcatctccagagaaaacaccaagaacacgctgtat ctgtagatgaacagcctgagtgccaaggacacggcactatatccctgtgcgaggaa

SEQ ID NO. 83 IGHV3-80 (F)

>IGHV3-80*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctgggggagatctggtgaagcctgggggatccctgagaetc tcttgtgtggcctctggattcaccttcagtagctactacatggaatgggtccgccaggct ccagggaaggggctgcagtgggtcgcacagattagcagtgatggaagtagcacatactac ccagacgctgtgaagggtcaattcaccatctccagagacaatgccaagaacacgctgtat ctgcagatgaacagcctgggagccgaggacacggccgtgtattactgtgcaaagga

SEQ ID NO. 84 IGHV3-81 (F)

>IGHV3-81*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctggaggaaacctggtgaagcctggggggtccctgagaetc tcttgtgtggcctctggattcaccttcagtagctactacatggactgggtccgccaggct ccagggaagaggctgcagtgggtcgcagggattagcagtgatggaagtagcacatactac ccacaggctgtgaagggccgattcaccatctccagagacaacgccaagaacacgctctat ctgcagatgaacagcctgagagccgaggactctgctgtgtattactgtgcgatgga

SEQ ID NO. 85 IGHV3-82 (F)

>IGHV3-82*01 I Canis lupus familiaris boxer | F | V-REGION | gaggtgcagctggtggagtctggaggagacctggtgaagtctggggggtccctgagaetc tcttgtgtggcctctggattcaccttcagtagctactacatgcactgggtccgccaggct acagggaaggggctgcagtgggtcacaaggattagcaatgatggaagtagcacaaggtac gcagacgccatgaagggccaatttaccatctccagagacaattccaagaatacgctgtat ctgcagatgaacagccagagagccgaggacatggccctatattactgtgcaaggga

SEQ ID NO. 86 IGHV3-83 (P)

>IGHV3-83*01 I Canis lupus familiaris boxer | P | V-REGION | gagttgcagctggtagagtctgggggagacctggtgaagcctggggggtctctgagactt tcttgtgtgtcctctggattcaccttcagtagctactggatgcactgggtcctccaggct ccagggaaagggctggagtgggtcgcaattattaacagtggtggaggtagcatatactac gcagacacagtgaagggccgattcaccatctccagagaaaacgccaagaacacgctctat ctgcagatgaacagcctgagagctgaggacagggccatgcattactgtgcgaaggga

SEQ ID NO. 87 IGHV4-1 (F)

>IGHV4-1* 01 I Canis lupus familiaris boxer | F | V-REGION | gaactcacactgcaggagtcagggccaggactggtgaagccctcacagaccctctctctc acctgtgttgtgtccggaggctccgtcaccagcagttactactggaactggatccgccag cgccctgggaggggactggaatggatggggtactggacaggtagcacaaactacaacccg gcattccagggacgcatctccatcactgctgacacggccaagaaccagttctccctgcag ctgagctccatgaccaccgaggacacggccgtgtattactgtgcaagaga SEQ ID NO. 88 IGHV(II) -1 (P)

>IGHV ( II ) -1*01 I Canis lupus familiaris boxer | P | V-REGION | ctggcacccctgcaggagtctgtttctgggctggggaaacccaggcagatccttacactc acctgctccttctctgggttcttattgagcatgtcagtatgggtgtcacatgggtccttt acccaccaggggaaggcactggagtcaatgccacatctggtgggagaacgctaagtacca cagcctgtctctgaacagcagcaagatgtatagaaagtccaacacttggaaagataaagg attatgtttcacaccagaagcacatctattcaacctgatgaacagccagcctgat

SEQ ID NO. 89 IGHV(II) -2 (P)

>IGHV ( 11 ) * 01 I Canis lupus familiaris boxer | P | V-REGION | ctggcacccctgcaggagtctgtttctgggctggggaaacccaggcagacccttacactc acctgctccttctctgggttcttattgagcatgtcagtgtgggtgtcacatgggtccttt acccaccaggggaaggcactggagtcaatgccacgtctggtgggagaacactaagtacca cagcctgtctctgaacagcagcaagatgtatagaaagtccaacacttggaaagataaagg attatgtttcacaccagaagcacatctattcaacctgatgaacaatcagcctgatgaga

Germline D sequences

SEQ ID NO. 90 IGHD1 (F)

>IGHD1* 01 I Canis lupus familiaris boxer | F | D-REGION | gtactactgtactgatgattactgtttcaac

SEQ ID NO. 91 IGHD2 (F)

>IGHD2* 01 I Canis lupus familiaris boxer | F | D-REGION | ctactacggtagctactac

SEQ ID NO. 92 IGHD3 (F)

>IGHD3* 01 I Canis lupus familiaris boxer | F | D-REGION | tatatatatatggatac

SEQ ID NO. 93 IGHD4 (F)

>IGHD4* 01 I Canis lupus familiaris boxer | F | D-REGION | gtatagtagcagctggtac

SEQ ID NO. 94 IGHD5 (ORF)

>IGHD5* 01 I Canis lupus familiaris boxer | ORF | D-REGION | agttctagtagttggggct

SEQ ID NO. 95 IGHD6 (F)

>IGHD6* 01 I Canis lupus familiaris boxer | F | D-REGION | ctaactggggc

Germline JH sequences

SEQ ID NO. 96 IGHJ1 (ORF)

>IGHJl* 01 I Canis lupus familiaris boxer | ORF | J-REGION | tgacatttactttgacctctggggcccgggcaccctggtcaccatctcctcag

SEQ ID NO. 97 IGHJ2 (F)

>IGHJ2* 01 I Canis lupus familiaris boxer | F | J-REGION | aacatgattacttagacctctggggccagggcaccctggtcaccgtctcctcag

SEQ ID NO. 98 IGHJ3 (F)

>IGHJ3* 01 I Canis lupus familiaris boxer | F | J-REGION | caatgcttttggttactggggccagggcaccctggtcactgtctcctcag

SEQ ID NO. 99 IGHJ4 (F)

>IGHJ4* 01 I Canis lupus familiaris boxer | F | J-REGION | ataattttgactactggggccagggaaccctggtcaccgtctcctcag SEQ ID NO. 100 IGHJ5 (F)

>IGHJ5* 01 I Canis lupus familiaris boxer | F | J-REGION | acaactggttctactactggggccaagggaccctggtcactgtgtcctcag

SEQ ID NO. 101 IGHJ6 (F)

>IGHJ6* 01 I Canis lupus familiaris boxer | F | J-REGION |

attactatggtatggactactggggccatggcacctcactcttcgtgtcctcag

Table 2. Canine IgK Sequence Information

Germline VK sequences

SEQ ID NO. 102 IGKV2-4 (F)

>IGKV2-4* 01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagacgccaccgtccctgtctgtcagccctagagagacggcctcc atctcctgcaaggccagtcagagcctcctgcacagtgatggaaacacctatttggattgg tacctgcaaaagccaggccagtctccacagcttctgatctacttggtttccaaccgcttc actggcgtgtcagacaggttcagtggcagcgggtcagggacagatttcaccctgagaatc agcagagtggaggctaacgatactggagtttattactgcgggcaaggtacacagcttcct cc

SEQ ID NO. 103 IGKV2-5 (F)

>IGKV2-5* 01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagaccccactgtccctgtccgtcagccctggagagccggcctcc atctcctgcaaggccagtcagagcctcctgcacagtaatgggaacacctatttgtattgg ttccgacagaagccaggccagtctccacagcgtttgatctataaggtctccaacagagac cctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgatgatgctggagtttattactgcgggcaaggtatacaagatcct cc

SEQ ID NO. 104 IGKV2-6 (F)

>IIGKV2-6*01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctcc atctcctgcaaggccagtcagagcctcctgcacagtgatggaaacacgtatttgaactgg ttccgacagaagccaggccagtctccacagcgtttaatctataaggtctccaacagagac cctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatactggagtttattactgcgggcaaggtatacaagatcct cc

SEQ ID NO. 105 IGKV2-7 (F)

>IGKV2-7* 01 I Canis lupus familiaris boxer | F | V-REGION | | gatattgtcatgacacagaacccactgtccctgtccgtcagccctggagagacggcctcc atctcctgcaaggccagtcagagcctcctgcacagtaacgggaacacctatttgaattgg ttccgacagaagccaggccagtctccacagggcctgatctataaggtctccaacagagac cctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatgctggagtttattactgcatgcaaggtatacaagctcct cc

SEQ ID NO. 106 IGKV2-8 (F)

>IGKV2- 8* 01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagaccccaccgtccctgtccgtcagccctggagagccggcctcc atctcctgcaaggccagtcagagcctcctgcacagtaacgggaacacctatttgaattgg ttccgacagaagccaggccagtctccacagggcctgatctatagggtgtccaaccgctcc actggcgtgtcagacaggttcagtggcagcgggtcagggacagatttcaccctgagaatc agcagagtggaggctgacgatgctggagtttattactgcgggcaaggtatacaagatcct cc SEQ ID NO. 107 IGKV2-9 (F)

>IGKV2- 9* 01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctcc atctcttgcaaggccagtcagagcctcctgcacagtgatggaaacacgtatttgaattgg ttccgacagaagccaggccagtctccacagcgtttgatctataaggtctccaacagagac cctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatactggagtttattactgcgggcaagttatacaagatcct cc

SEQ ID NO. 108 IGKV2-10 (F)

>IGKV2-10*01 I Canis lupus familiaris boxer | F | V-REGION | gatattgtcatgacacagaccccactgtccctgtccgtcagccctggagagactgcctcc atctcctgcaaggccagtcagagcctcctgcacagtgatggaaacacgtatttgaattgg ttccgacagaagccaggccagtctccacagcgtttgatctataaggtctccaacagagac cctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatactggagtttattactgcatgcaaggtacacagtttcct eg

SEQ ID NO. 109 IGKV2-11 (F)

>IGKV2-11*01 I Canis lupus familiaris boxer | F | V-REGION | gatatcgtcatgacacagaccccactgtccctgtccgtcagccctggagagactgcctcc atctcctgcaaggccagtcagagcctcctgcacagtaacgggaacacctatttgttttgg ttccgacagaagccaggccagtctccacagcgcctgatcaacttggtttccaacagagac cctggggtcccacacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatgctggagtttattactgcgggcaaggtatacaagctcct cc

SEQ ID NO. 110 IGKV2S12 (P)

>IGKV2S12*01 I Canis lupus familiaris boxer | P | V-REGION | gatatcgtgatgacccagaccccattgtccttgcctgtcacccctggagagctagcctca tcactgtgcaggaggccagtcagagcctcctgcacagtgatggatatatttatttgaatt ggtactttcagaaatcaggccagtctccatactcttgatctatatgctttacaaccagac ttctggagtcccaggctggttcattggcagtggatcagggacagatttcaccctgaggat cagcagggtggaggctgaagatgctggagtttattattgccaacaaactctacaaaatcc tee

SEQ ID NO. 111 IGKV2S13 (F)

>IGKV2S13*01 I Canis lupus familiaris boxer | F | V-REGION | gatatcgtcatgacgcagaccccactgtccctgtctgtcagccctggagagccggcctcc atctcctgcagggccagtcagagcctcctgcacagtaatgggaacacctatttgtattgg ttccgacagaagccaggccagtctccacagggcctgatctacttggtttccaaccgtttc tcttgggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaate agcagagtggaggctgacgatgctggagtttattactgcgggcaaaatttacagtttcct tc

SEQ ID NO. 112 IGKV2S14 (P)

>IGKV2S14*01 I Canis lupus familiaris boxer | P | V-REGION | gaggttgtgatgatacagaccccactgtccctgtctgtcagccctggagagccggcctcc atctcctgcagggccagtcagagtctccggcacagtaatggaaacacctatttgtattgg facetgcaaaagccaggccagtctccacagcttctgatcgacttggtttccaaccatttc actggggtgtcagacaggttcagtggcagcgggtctggcacagattttaccctgaggatc agcagggtggaggctgaggatgttggagtttattactgcatgcaaagtacacatgatcct cc

SEQ ID NO. 113 IGKV2S15 (P)

>IGKV2S15*01 I Canis lupus familiaris boxer | P | V-REGION | gatatcatgatgacacagaccccactctccctgcctgccacccctggggaattggctgcc atcttctgcagggccagagtctcctgcacaataatggaaacacttatttacactggttcc tgcagacatcaggccaggttccaaggcatctgaaccatttggcttccagctgttactctg gggtctcagacaggttcagtggcaacgggtcagggacagatttcacactgaaaatcagca gagtggaggctgaggatgttagtgtttattagtgcctgcaagtacacaaccttccatc SEQ ID NO. 114 IGKV2S16 (F)

>IGKV2S16*01 I Canis lupus familiaris boxer | F | V-REGION | gaggccgtgatgacgcagaccccactgtccctggccgtcacccctggagagctggccact atctcctgcagggccagtcagagtctcctgcgcagtgatggaaaatcctatttgaattgg tacctgcagaagccaggccagactcctcggccgctgatttatgaggcttccaagcgtttc tctggggtctcagacaggttcagtggcagcgggtcagggacagatttcacccttaaaatc agcagggtggaggctgaggatgttggagtttattactgccagcaaagtctacattttcct cc

SEQ ID NO. 115 IGKV2S18 (P)

>IGKV2S18*01 I Canis lupus familiaris boxer | P | V-REGION | gatatcgtcatgacacagaccccactgtccgtgtctgtcagccctggagagacggcctcc atctcctgcagggccagtcagagcctcctgcacagtgatggaaacacctatttggattgg facetgcagaagccaggccagattccaaaggacctgatctatagggtgtccaactgcttc actggggtgtcagacaggttcagtggcagcgggtcagggacagatttcaccctgagaatc agcagagtggaggctgacaacgctggagtttattactgcatgcaaggtatacaagatcct cc

SEQ ID NO. 116 IGKV2S19 (F)

>IGKV2S19*01 I Canis lupus familiaris boxer | F | V-REGION | gatatcgtcatgacacagactccactgtccctgtctgtcagccctggagagacggcctcc atctcctgcagggccaatcagagcctcctgcacagtaatgggaacacctatttggattgg tacatgcagaagccaggccagtctccacagggcctgatctatagggtgtccaaccacttc actggcgtgtcagacaggttcagtggcagcgggtcagggacagatttcaccctgaagatc agcagagtggaggctgacgatgctggagtttattactgcgggcaaggtacacactctcct cc

SEQ ID NO. 117 IGKV3-3 (P)

>IGKV3-3* 01 I Canis lupus familiaris boxer | P | V-REGION | gaaatagtcttgacctagtctccagcctccctggctatttcccaaggggacagagtcaac catcacctatgggaccagcaccagtaaaagctccagcaacttaacctggtaccaacagaa ctctggagcttcttctaagctccttgtttacagcacagcaagcctggcttctgggatccc agctggcttcattggcagtggatgtgggaactcttcctctctcacaatcaatggcatgga ggctgaaggtgctgcctactattactaccagcagtagggtagctatctgct

SEQ ID NO. 118 IGKV3 S 1 (F)

>IGKV3S1* 01 I Canis lupus familiaris boxer | F | V-REGION | gaaatcgtgatgacacagtctccagcctccctctccttgtctcaggaggaaaaagtcacc atcacctgccgggccagtcagagtgttagcagctacttagcctggtaccagcaaaaacct gggcaggctcccaagctcctcatctatggtacatccaacagggccactggtgtcccatcc cggttcagtggcagtgggtctgggacagacttcagcttcaccatcagcagcctggagcct gaagatgttgcagtttattactgtcagcagtataatagcggatata

SEQ ID NO. 119 IGKV3 S2 (P)

>IGKV3S2* 01 I Canis lupus familiaris boxer | P | V-REGION | gagattgtgccaacctagtctctagccttctaagactccagaagaaaaagtcaccatcag ctgctgggcagtcagagtgttagcagctacttagcctggtaccagcaaaaacctggacag gctcccaggctcttcatctatggtgcatccaacagggccactggtgtcccagtccgcttc agcggcagtgggtgtgggacagatttcaccctcatcagcagcagtctggagtcagtctga agatgttgcaacatattactgccagcagtataatagctacccacc

SEQ ID NO. 120 IGKV4S1 (F)

>IGKV4S1* 01 I Canis lupus familiaris boxer | F | V-REGION | gaaatcgtgatgacccagtctccaggctctctggctgggtctgcaggagagagcgtctcc atcaactgcaagtccagccagagtcttctgtacagcttcaaccagaagaactacttagcc tggtaccagcagaaaccaggagagcgtcctaagctgctcatctacttagcctccagctgg gcatctggggtccctgcccgattcagcagcagtggatctgggacagatttcaccctcacc atcaacaacctccaggctgaagatgtgggggattattactgtcagcagcattatagttct cctcc SEQ ID NO. 121 IGKV4-1 (ORF)

>IGKV4-1* 01 I Canis lupus familiaris boxer | ORF | V-REGION | gacatcacgatgactcagtgtccaggctccctggctgtgtctccaggtcagcaggtcacc acgaactgcagggccagtcaaagcgttagtggctacttagcctggtacctgcagaaacca ggacagcgtcctaagctgctcatctacttagcctccagctgggcatctggggtccctgcc cgattcagcagcagtggatctgggacagatttcaccctcaccgtcaacaacctcgaggct gaagatgtgagggattattactgtcagcagcattatagttctcctct

SEQ ID NO. 122 IGKV7-2 (P)

>IGKV7-2* 01 Canis lupus familiaris boxer V-REGION

gacattatgctgacccagtctccagcctccttgaccatgtgtctccaggagagaggg cca ccatctcttgcagggccagtcagaaagccagtgatatttggggcattacccaccatatta ccttgtaccaacagaaatcagaacagcatcctaaagtcctgattaatgaagcctccagtt gggtctggggtcctaggcaggttcagtggctgtgggtctgggactgatttcagcctcaca attgatcctgtggaggctggcgatgctgtcaactattactgccagcagagtaaggagtct cctcc

SEQ ID NO. 123 IGKV(II)-1 (P)

>IGKV ( II ) -1*01 I Canis lupus familiaris boxer | P | V-REGION | gaaattgcagattgtcaaatggataataccaggatgcggtctctagcctccctgactccc aggggagagaaccatcattacccataaaataaatcctgatgacataataagtttgcttgg tatcaatagaaaccaggtgagattcctcgagtcctggtatacgacacttccatccttaca ggtcccaaactggttcagtggcagtgtctccaagtcagatcttactctcatcatcagcaa tgtgggcacacctgatgctgctacttattactgttatgagcattcagga

Germline JK sequences

SEQ ID NO. 124 IGKJ1 (F)

>GKJ1*01 I Canis lupus familiaris boxer | F | J-REGION |

gtggacgttcggagcaggaaccaaggtggagctcaaac

SEQ ID NO. 125 IGKJ2 (ORF)

>IGKJ2* 01 I Canis lupus familiaris boxer | ORF | J-REGION | tttatactttcagccagggaaccaagctggagataaaac

SEQ ID NO. 126 IGKJ3 (F)

>IGKJ3* 01 I Canis lupus familiaris boxer | F | J-REGION | gttcacttttggccaagggaccaaactggagatcaaac

SEQ ID NO. 127 IGKJ4 (F)

>IGKJ4* 01 I Canis lupus familiaris boxer | F | J-REGION | gcttacgttcggccaagggaccaaggtggagatcaaac

SEQ ID NO. 128 IGKJ5 (F)

>IGKJ5* 01 I Canis lupus familiaris boxer | F | J-REGION | gatcacctttggcaaagggacacatctggagattaaac

Table 3. Canine Iel Sequence Information

Germline nl sequences

SEQ ID NO. 129 IGLV1-35 (P)

>IGLV1-35*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagctggcctcggtgtctggggccctgggccacagggtcagcatc tcctggactggaagcagctccaacataagggttgattatcctttgagctgataccaacag ctcccagaatgaagaacgaacccaaactcctcatctatggtaacagcaattggctctcag gggttccagatccattctctagaggctccaagtctggcacctcaggctccctgaccaact ctggcctccaggctgaggacgaggctgattgttactgcgcagcgtgggacatggatctca gtgctc

SEQ ID NO. 130 IGLV1-37 (ORF)

>IGLV1-37*01 I Canis lupus familiaris boxer | ORF | V-REGION | caatctgtgctgactcagctggcctcagtgtctgggtccttgggccagagggtcaccatc tcctgctctggaagcacaaatgacattggtattattggtgtgaactggtaccagcagctc ccagggaaggcccctaaactcctcatatacgataatgagaagcgaccctcaggtatcccc gatcgattctctggctccaagtctggcaactcaggcaccctgaccatcactgggctccag gctgaggacgaggctgattattactgccagtccatggatttcagcctcggtggt

SEQ ID NO. 131 IGLV1-41 (ORF)

>IGLV1-41*01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctgactcagccagcctccgtgtctgggtccctgggccagagggtcaccatt tcctgcactggaagcagctccaacgttggttatagcagtagtgtgggctggtaccagcag ttcccaggaacaggccccagaaccatcatctattatgatagtagccgaccctcgggggtc cccgatogattctctggctccaagtctggcagcacagccaccctgaccatctctgggetc caggctgaggatgaggctgattattactgctcatcttgggacaacagtctcaaagctcc

SEQ ID NO. 132 IGLV1-44 (F)

>IGLV1-44*01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgaatcagccggcctcagtgtctggggccctgggccagaaggtcaccatc tcctgctctggaagcacaaatgacattgatatatttggtgtgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtggacagtgatggggatcgaccctcaggggtccct gacagattttctggctccagctctggcaactcaggcaccctgaccatcactgggctccag gctgaggacgaggctgattattactgtcagtctgttgattccacgcttggtgctca

SEQ ID NO. 133 IGLV1-45 (P)

>IGLV1-45*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtactgactcaatcagcctcagcgtctgggtccttgggccagagggtctccgtc tcctgctctagcagcacaaacaacattggtattattggtgtgaagtggtaccagcagatc ccaagaaaggcccctaaactcctcatatatgataatgagaagagaccctcaggtgtcccc aattgattctctggctccaagtctggcaacttaggcaccctaaccatcaatgggcttcag gctgagggcgaggctgattattactgccagtccatggatttcagcctcggtggt

SEQ ID NO. 134 IGLV1-46 (F)

>IGLV1-46*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcaaccagcctcagtgtccgggtctctgggccagagggtcaccatc tcctgcactggaagcagctccaacattggtagagattatgtgggctggtaccaacagctc ccgggaacacgccccagaaccctcatctatggtaatagtaaccgaccctcgggggtcccc gatcgattctctggctccaagtcaggcagcacagccaccctgaccatctctgggctccag gctgaggacgaggctgattattactgctctacatgggacaacagtctcactgttcc

SEQ ID NO. 135 IGLV1-48 (F)

>IGLV1-48*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctatgctgactcagccagcctcagtgtctgggtccctgggccagaaggtcaccatc tcctgcactggaagcagctccaacatcggtggtaattatgtgggctggtaccaacagctc ccaggaataggccctagaaccgtcatctatggtaataattaccgaccttcaggggtcccc gatcgattctctggctccaagtcaggcagttcagccaccctgaccatctctgggctccag gctgaggacgaggctgagtattactgctcatcatgggatgatagtctcagaggtca

SEQ ID NO. 136 IGLV1-49 (F)

>IGLV1-49*01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactcagccgccctcagtgtctgcggtcctgggacagagggtcaccatc tcctgcactggaagcagcaccaacattggcagtggttatgatgtacaatggtaccagcag ctcccaggaaagtcccctaaaactatcatctatggtaatagcaatcgaccctcaggggtc ccggatcgcttctctggctccaagtcaggcagcacagcctctctgaccatcactgggctc caggctgaggacgaggctgattattactgccagtcctctgatgacaacctcgatgatca

SEQ ID NO. 137 IGLV1-50 (P)

>IGLV1-50*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccggcctca ... gtgtccgggtctctgggccagagagtcacc atctcctgcactggaagcagctccaacatc . gatagaaaatat gttggctggtaccaacagctc ... ccgggaacaggccccagaaccgtcatctatgataat

. agtaaccgaccctcgggggtccct ... gatcgattctct ggctccaag . tcaggcagcacagccaccctgaccatctctgggctccaggctgag gacgaggctgat ... tattactgctcaacatacgacagcagtctcagtagtgg

SEQ ID NO. 138 IGLV1-52 (P)

>IGLV1-52*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcagtagatataatgtgaactggtaccaacagctc ctgggaacaggccccagaaccctcatctatggtagtagtaaccgaccctcgggggtcccc gattgattctctggctccaagtcaggcagcccagctaccctgaccatctctgggctccag gctgaggatgaggctgattattactgctcaacatacgacaggggtctcagtgctcg

SEQ ID NO. 139 IGLV1-54 (P)

>IGLV1-54*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgactcagccgccctcagggtctgggggcctgggccagaggttcagcatc tcctgttctggaagcacaaacaacatcagtgattattatgtgaactggtactaacagctc ccagggacagcccctaaaaccattatctatttggatgataccagaccccctggggtcccg gattgattctctgtctccaagtctagcagctcagctaccctgaccatctctgggctccag gctgaggatgaagctgattattactgctcatcctggggtgatagtctcaatgctcc

SEQ ID NO. 140 IGLV1-55 (F)

>IGLV1-55*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagaggatcaccatc tcctgcactggaagcagctccaacattggaggtaataatgtgggttggtaccagcagctc ccaggaagaggccccagaactgtcatctatagtacaaatagtcgaccctcgggggtgccc gatcgattctctggctccaagtctggcagcacagccaccctgacoatctctgggctccag gctgaggatgaggctgattattactgctcaacgtgggatgatagtctcagtgctcc

SEQ ID NO. 141 IGLV1-56 (ORF)

>IGLV1-56*01 I Canis lupus familiaris boxer | ORF | V-REGION | cggtctgtgctgactcagccgccctcagtgtcgggatctgtgggccagagaatcaccatc tcccgctctggaagcacaaacagcattggtatacttggtgtgaactggtaccaagagctc ccaggaaaggcccctaaactcctcgtagatggtactgggaatagaccctcaggggtccct gaccgattttctggctccaaatctggcaactcaggcactctgaccatcactgggcttcag cctgaggacgaggctgattattattgtcagtccattgaacccatgcttggtgctcc

SEQ ID NO. 142 IGLV1-57 (F)

>IGLV1-57*01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactccgctgccctcagtgtctgcggccctgggacagacggtcaccatc tcttgtactggaaatagcacccaaatcagcagtggttatgctgtacaatggtaccagcag ctcccaggaaagtcccctgaaactatcatctatggtgatagcaatcgaccctcgggggtc ccagatogattctctggcttcagctctggcaattcagccacactggccatcactgggetc caggatgaggacgaggctgattattactgccagtccttagatgacaacctcaatggtca

SEQ ID NO. 143 IGLV1-58 (F)

>IGLV1-58*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagatatagtgttggctggttccagcagctc ccgggaaaaggccccagaaccgtcatctatagtagtagtaaccgaccctcaggggtccct gatcgattctctggctccaagtcaggcagcacagccaccctgaccatctctgggctccag gctgaggacgaggctgattattactgctcaacatacgacagcagtctcagtagtag

SEQ ID NO. 144 IGLV1-61 (P)

>IGLV1-61*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgacatagccaccctcagtgtctggggccctgggccagagggtcaccatc tcctgcactggaagcagctcaagcatgggtagttattatgtgagctggcacaagcagctc ccaggaacaggccccagaaccatcatgtgttgtaaaaacatcgaccttcgggaatctcca atcaagtctctggctcccattctggcaacacagccaccctgaccatcactgggctcctgg ctgaggatgaggctgattattactgttcaacatgggatgacaatctcaatgcacc SEQ ID NO. 145 IGLV1-63 (P)

>IGLV1-63*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagctgccctcagtgtctggggccctgggccagagggtcaccatc tcctgctctggaagcagctctaaacttggggcttatgctctgaactagaaccaacaattc ccaggaacagattccaatttcctcatctatgatgatagtaattgatctttctggatgcct gattaattctgtggctccacatccagcagttcaggctccctgaccatcactgggctctgg gatgaggacaaggctgattattactgccagtgccattaccatagcctccgtgct

SEQ ID NO. 146 IGLV1-65 (P)

>IGLV1-65*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccagcctcagtgtctggatccctgggccaaagggtcaccatc tcctgcactggaagcacaaacaacatcggtggtgataattatgtgcactggtaccaacag ctcccaggaaaggcacccagtctcctcatctatggtgatgataacagagaatctggggtc ccggaacgattctctggctccaagtcaggcagctcagccactctgaccatcactgggctc catgctgaggacgaggctgatattattgccagtcctacgatgacagcctcaatactca

SEQ ID NO. 147 IGLV1-66 (F)

>IGLV1-66*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccgccctcagtgtcaggatctgtgggccagagaatcaccatc tcctgctctggaagcacaaacagcattggtatacttggtgtgaactggtaccaactgctc tcaggaaaggcccctaaactcctcgtagatggtactggaaatcgaccctcaggggtccct gaccgattttctggctccaaatctggcaactcaggcactctgaccatcactgggcttcag cctgaggacgaggctgattattattgtcagtccattgaacccatgcttggtgctcc

SEQ ID NO. 148 IGLV1-67 (F)

>IGLV1-67*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtcctgactcagccggcctcagtgtctggggttctgggccagagggtcaccatc tcctgcactggaagcagctccaacattggtggaaattatgtgagctggcaccagcaggtc ccagaaacaggccccagaaacatcatctatgctgataactaccgagcctcgggggtccct gatcgattctctggctccaagtcaggcagcacagccaccctgacoatctctgtgotocag gctgaggatgaggctgattattactgctcagtgggggatgatagtctcaaagcacc

SEQ ID NO. 149 IGLV1-68 (P)

>IGLV1-68*01 I Canis lupus familiaris boxer | P | V-REGION | cagtccatcctgactcagcagccctcagtctctgggtcactgggccagagggtcaccatc tcttgcactggattccctagcaacaatgattatgatgcaatgaaaattcatacttaagtg ggctggtaccaacagtccccaggaaagtcacccagtctcctcatttatgatgaaaccaga aactctggggtccctgatcgattctctggctccagaactggtagctcagcctccctgccc atctctggactccaggctgaggacaagactgagtattactgctcagcatgggatgatcgt cttgatgctca

SEQ ID NO. 150 IGLV1-69 (P)

>IGLV1-69*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctaactcagccaccctcagtgtcggggtcgctgggccagagggtcaccatc tcctgctctggaagcacaaacaacatcagtattgttggtgcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtggacagtgatggggatcgaccgtcaggggtccct gaccgattttctggctctaagtctggcaaatcagccaccctgacoatcactgggcttcag gctgaggacgaggctgattattactgtatattggtcccacgctttgtgctca

SEQ ID NO. 151 IGLV1-69-1 (P)

>IGLVl-69-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccactgttagggcctggggccctgggcagagggtcaccctct cctgacctggaagagtcccagtattggtgattatggtatgaaatggtacaagcagcttgc aaggacagaccccagactcgtcatctatggcaatagcaattgatcctcgggtccccaatc aattttctggctctggttttggcatcactggctccttgaccacctctgggctccagactg aaaaataggctgattactagtgcttctccagtgatccaggcctgt

SEQ ID NO. 152 IGLV1-70 (F)

>IGLV1-70*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcaaccggcctccgtgtctgggtccctgggccagagagtcaccatc tcttgcactagaagcagctcgaacgttggctatggcaatgatgtgggatggtaccagcag ctcccaggaacaggccccagaaccatcatctataataccaatactcgaccctctggggtt cctgatcgattctctggctccaaatcaggcagcacagccaccctgaccatctctggactc caggctgaggacgaggctgattattactgctcttcctatgacagcagtctcaatgctca

SEQ ID NO. 153 IGLV1-72 (ORF)

>IGLV1-72*01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctaactcagccggcctcagtgtctggttccctgggtcagagggtcaccatc tgcactggaagcagctccaacattggtacatatagtgtaggctggtaccaacagctccca ggatcaggccccagaaccatcatctatggtagtagtaaccgaccgttgggggtccctgat cgattctctggctccaggtcaggcagcacagccaccctgaccatctctgggctccaggct gaggacgaagctgattattactgcttcacatacgacagtagtctcaaagctcc

SEQ ID NO. 154 IGLV1-73 (F)

>IGLV1-73*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgaatcagccaccttcagtgtctggatccctgggccagagaatcaccatc tcctgctctggaagcacgaatgacatcggtatgcttggtgtgaactggtaccaacagctc ccaggaaatgcccctaaactccttgtagatggtactgggaatcgaccctcaggggtccct gaccaattttctggctccaaatctggcaattcaggcactctgaccatcactgggctccag gctgaggacgaggctgattattattgtcagtcctatgatctcacgcttggtgctcc

SEQ ID NO. 155 IGLV1-74 (P)

>IGLV1-74*01 I Canis lupus familiaris boxer | P | V-REGION | | cagtccatgatgactcagccaccctcagtgtctgggtcactgggccagagggtcaccatc tactgcactggaatccctagcaacactgattatagtggattggaaatttatacttatgtg agctggtaccaacagtataaggaaaggcacccagtctcctcatctatggggatgataccg gaaactctgaggtccctgatcaattctctggctccaggtctggtagctcaacctccctga ccatctctggactccaggctgaggatagtcttaatgctca

SEQ ID NO. 156 IGLV1-75 (F)

>IGLV1-75*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgactgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtggatataatgttggctggttccagcagctc ccgggaacaggccccagaaccgtcatctatagtagtagtaaccgaccctcgggggtcccg gatcgattctctggctccaggtcaggcagcacagccaccctgacoatctctgggctccag gctgaggacgaggctgagtattactgctcaacatgggacagcagtctcaaagctcc

SEQ ID NO. 157 IGLV1-78 (P)

>IGLV1-78*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccggcctcagtgtccaggtccctgggccagatagtcaccatc tcttgcgctggaagcagctccaacatccgtacaaaatatgtgggctggtactaacagctc ccgagaacaggccccagaaccgtcatctatggtaatagtaactgaccctcgggggtcctc gatcaattctctggctccaagtcaggcagcatagccaccctgacoatctctgtgotocag gctgaggacgaggcttattattactgctcaacatatgacagcagtctcagtgctct

SEQ ID NO. 158 IGLV1-79 (P)

>IGLV1-79*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccggcctctgtgtctggggccctgggccagaggtcaecatct cctgcactaggagcagctccaatgttggttatagcagttatgtgggctggtaccagcagc tcccaggaacaggccccaaaaccatcatctataataccaatactcgaccctctggggttc ctgatcgattctctggctccaaatcaggcagcacagccacccttaccattgctggactcc aggctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc

SEQ ID NO. 159 IGLV1-79-1 (P)

> IGLVl-79-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctatgctgactcaccctggccagaggatcaccctctcctgacctggaagagtccca gtattggtgattatggtgtgaaatggtacaggcagctagcaagaacagaccccagactcc tcatttatagcaatagcaatcgatccttgagtccccaatcaattttccgcctctggtttt gacattactggctccttgaccacctccaggctccagactgaaaaataggctgattactag tgcttatacagtgatccaggcttgtggggctg SEQ ID NO. 160 IGLV1-80 (F)

>IGLV1-80*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccgacctcagtgtcgtggtccctgggccagagggtcacaatc tcatgctctagaagcacgaataacatcggtattgtcggggcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtggacagtgatggggatcaactgtcaggggtccct gaccgattttctggctccaagtctggcaactcagccaacctgaccatcactgggctccag gctgaggacaaggctgattattactgccagtcctttgatcacacgcttggtgctcg

SEQ ID NO. 161 IGLV1-81 (P)

>IGLV1-81*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgttgagtcagccagcctcagtgtctggggttctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtggaaattacgtgagctggcaccagcaggtc ccagaaacaggccccagaaacatcatctatgctgataactactgagcctcgggggtccct gatggattctctggctccaagtaaggcagcacagccaccccgacoatctctgtgotocag gctgaggatgaggctgattattactgctcagtgggggataatagtctcaaagcacc

SEQ ID NO. 162 IGLV1-82 (F)

>IGLV1-82*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccagcctcagtgtcggggtccctgggccagagagtcaccatc tcctgctctggaaggacaaacatcggtaggtttggtgctagctggtaccaacagctccca ggaaaggcccctaaactcctcgtggacagtgatggggatcgaccgtcaggggtccctgac cgattttccggctccaagtctggcaactcggccactctgaccatcactggtctccatgct gaggacgaggctgattattactgtctgtctattggtcccacgcttggtgctca

SEQ ID NO. 163 IGLV1-82-1 (P)

>IGLVl-82-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccactgttagggcctggggccctggccagaggctcactctct cctgccctggaagagtcccagtattggtgattatgatgtgaagtggtacaggcagctcac aagaacagaccctagactcctcatccatggtgatagcaattgatcctcgggtccccaatc acttttctggctctgtttttggcatcactggctgcttgaccacctctgggctccagactg aaaaataggctgattactagtgcttatccagtgatccag

SEQ ID NO. 164 IGLV1-83 (P)

>IGLV1-83*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccggcctctgtgtctggggccctgggccagaggtcaecatct cctgcactaggagcagctccaatgttggttatagcagttatgtgggctggtaccagcagc tcccaggaacaggccccaaaaccatcatctataataccaatactcgaccctctggggtcc ctgatcgattctctggctccaaatcaggcaggacagccacccttaccattgctggactcc aggctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc

SEQ ID NO. 165 IGLV1-84 (F)

>IGLV1-84*01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaatgttggttatggcaattatgtgggctggtaccagcag ctcccaggaacaggccccagaaccctcatctatggtagtagttaccgaccctcgggggtc cctgatcgattctctggctccagttcaggcagctcagccacactgaccatctctgggctc caggctgaggatgaagctgattattactgctcatcctatgacagcagtctcagtggtgg

SEQ ID NO. 166 IGLV1-84-1 (ORF)

>IGLVl-84-l*01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctgactcagccagcctcagcgtctgggtccttgggccagagggtcactgtc tcctgctctagcagcacaaacaacatcggtattattggtgtgaagtggtaccagcagatc ccaggaaaggcccataaactcctcatatatgataatgagaagcgaccctcaggtgtcccc aatcgattctctggctccaagtctggcgacttaagcaccctgaccatcaatgggcttcag ggtgaggacgaggctgattattattgccagtccatggatttcagcctcggtggtca

SEQ ID NO. 167 IGLV1-86 (ORF)

>IGLV1-86*01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctgactcagccagcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaatccccagcaacacagattttgatggaatagaatttgatacttctgtg agctggtaccaacagctcccagaaaagccccctaaaaccatcatctatggtagtactctt tcattctcgggggtccccgatcgattctctggctccaggtctggcagcacagccaccctg accatctctgggctccaggctgaggacgaggctgattattactgctcatcctgggatgat agtctcaaatcata

SEQ ID NO. 168 IGLV1-87 (F)

>IGLV1-87*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccagcctcagtgtctggatccctgggccaaagggtcaccatc tcctgcactggaagcacaaacaacatcggtggtgataattatgtgcactggtaccaacag ctcccaggaaaggcacccagtctcctcatctatggtgatgataacagagaatctggggtc cctgaacgattctctggctccaagtcaggcagctcagccactctgaccatcactgggctc caggctgaggacgaggctgattattattgccagtcctacgatgacagcctcaatactca

SEQ ID NO. 169 IGLV1-88 (P)

>IGLV1-88*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccgccctcagtgtcgggatctgtgggccagagaatcaccatc tcctgctctggaagcacaaacagctaccaacagctctcaggaaaggcctctaaactcctc gtagatggtactgggaaccgaccctcaggggtccccgaccgattttctggctccaaatct ggcaactcaggcactctgaccatcactgggcttgggacgaggctgaggacgaggctgagg acgaggctgattattattgttagtccactgatctcacgcttggtgctcc

SEQ ID NO. 170 IGLV1-88-2 (P)

>IGLVl-88-2*01 I Canis lupus familiaris boxer | P | V-REGION | caggccgccctgggcaatgagttcgtgcaggtcaaggctgagacagacctgcagaattca ggtttgtctgagacacagctcatcagatgtgtgcagtgtgtgtcctggtaccaacggctc ccatgaatgggtcctaaatccttatctagaaataacatttagatcactttgtggcccgga tccattctctggctccatgtctggcaactctggcctcatgaacatcactgggctatggtc tgaagatggagctgctcttcacaggccctcttgggacaaaattcttggggct

SEQ ID NO. 171 IGLV1-88-3 (P)

>IGLVl-88-3*01 I Canis lupus familiaris boxer | P | V-REGION | cagtccatcctgactcagccgccctcagtctctgggtcactgggccagagggtcaccatc tcctgcaatggaatccctgacagcaatgattatgatgcatgaaaattcatacttacgtga gctggtaccaacagttcccaagaaagtcaccagtctcctcatctacgatgataccagaaa ctctggggaccctgatcaattctctggctccagatctggtaactcagcctccctgcccat ctctggactccaggctgaggacgaggctgagtattactgctcagcatgggatgatcgtct tgatgctca

SEQ ID NO. 172 IGLV1-89 (P)

>IGLV1-89*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtactgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtggatattatgtgagctggctctagcagctc ccgggaacaggccccagaaccatcatctatagtagtagtaaccgaccttcaggggtccct gatcgattctctggctccaggtcaggcagcacagccaccctgaccatctctgggctccag gctgaggatgaggctgattattactgttcaacatacgacagcagtctcaaagctcc

SEQ ID NO. 173 IGLV1-89-2 (P)

>IGLVl-89-2*01 I Canis lupus familiaris boxer | P | V-REGION | cttcctgtgctgacccagccaccctcaaggtctgggggtctggttcagaagatcaccatc ttctgttctggaagcacaaacaacatgggtgataattatgttaactggtacaaacagctt ccaggaacggcccctaaaaccatcatctaagtggatcatatcagaccctcaggggtcctg gagagattctctgtctccaattctggcagctcagccaacctgaccatctctgggctccag gatgaggactaggctgattattattgctcatcctggcatgatagtctcagtgctcc

SEQ ID NO. 174 IGLV1-91 (P)

>IGLV1-91*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgctgactcagctgccctcagtgtctgcagccctgggacagagggtcaccatc tgcactggaagcagcaccaacatcggcagtggttattatacactatggtaccagcagctg caggaaagtcccctaaaactatcatctatggtaatagcaatcgacccttgagggtcccgg atcgattctctggctccaagtatggcaattcagccacgctgaccatcactgggctccagg ctgaggacgaggatgattattactgccagtcctctgatgacaacctcgatggtca SEQ ID NO. 175 IGLV1-92 (F)

>IGLV1-92*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcggtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaatgttggttatggcaattatgtgggctggtaccagcag cttccaggaacaggccccagaaccattatctgttataccaatactcgaccctctggggtt cctgatcgatactctggctccaagtcaggcagcacagccaccctgaccatctctgggctc caggctgaagacgagactgattattactgtactacgtgtgacagcagtctcaatgctag

SEQ ID NO. 176 IGLV1-94 (F)

>IGLV1-94*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagcctccctcagtgtccgggttcctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgcactggtaccaacagctc ccaggaacaggccccagaaccctcatctatggtattagtaaccgaccctcaggggtcccc gatcgattctctggctccaggtcaggcagcacagccactctgacaatctctgggctccag gctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 177 IGLV1-95-1 (P)

>IGLVl-95-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccactgttagggcctgggttcctggccagagggtcaccctct cctgccctggaagagtctcagttttggtgattatggtgtgaaacggtacaggaagctcgc atggacagaccccagactcctcatctatggcaatagcaattgattctcgggtccccagtc tattttctggctctggttttggcatcactggctccttgaccacctccgggctccagactg aaaaataggctgatttctagtgcttctccagtgatccaggccttt

SEQ ID NO. 178 IGLV1-96 (F)

>IGLV1-96*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgcgctgactcaaacggcctccatgtctgggtctctgggccagagggtcaccgtc tcctgcactggaagcagttccaacgttggttatagaagttatgtgggctggtaccagcag ctcccaggaacaggccccagaaccatcatctataataccaatactcgaccctctggggtt cctgatogattctctggctccatatcaggcagcacagccaccctgactattgctggaetc caggctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc

SEQ ID NO. 179 IGLV1-97 (P)

>IGLV1-97*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgaatcagctgccttcagtgttaggatccctgggccagagaatcaccatc tcctgctctggaagcacgaatgacatcggtatgcttggtgtgaactggtaccaagagccg ccaggaaaggcccctaaactcctcgtagatggtactgggaatcgaccctcagggtccctg ccgattttctggctccaaatctggcaactcaggcactctgaccatcactgggctccaggc tgaggacgaggctgattattattgtcagtccactgatctcacgcttggtgctcc

SEQ ID NO. 180 IGLV1-97-4 (F)

>IGLVl-97-4*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagcctccctcagtgttcaggtccctgggccagagggtcactata tcctgcactggaagcagctccaacgtcggtagaggttatgtgatctggtaccaacagctc ctgggaacacgcccaagaaccctcatatatggtagtagtaaccaaccctcaggggtcccc aatcaattctctggctccaggtcaggcagcacagacactctgacaatctctgggttccag gctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 181 IGLV1-98 (P)

>IGLV1-98*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccagtctcagtgtctggggccctgtgccagagggtcaccatc tcctgcactggaaacagctccaacattggttatagcagttgtgtgagctgatatcagcag ctcccaggaacaggccccagaaccatcatctatagtatgaatactcaaccctctggggtt cctgatcgattctctggctccaggtcaggcaactcagccaccctaaccatctctgggctc caggctgaggacaaggctgactattactgctcaacatatgacagcagtctcagtgctca

SEQ ID NO. 182 IGLVl-100 (F)

> IGLV1-100*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccgacctcagtgtcggggtcccttggccagagggtcaccatc tcctgctctggaagcacgaacaacatcggtattgttggtgcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtggacagtgatggggatcgaccgtcaggggtccct gaccggttttccggctccaagtctggcaactcagccaccctgaccatcactgggcttcag gctgaggacgaggctgattattactgccagtcctttgataccacgcttgatgctca

SEQ ID NO. 183 IGLVl-100-1 (P)

>IGLV1-100-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtactgactcagcagccgttagtgcttggggccctggccagagggtcagcttct cctgccttggaagagtcccagtattggtaattatggtgtgaaatggtacaagcagctcaa aaggacagaccccagacttctcatctatggcaatagcaattgatcctcgggtccccaatc aattttctggctctggttttggcatcactggctccttgaccacctatgggctccagactg aaaaataggctgattactagtgcttttccagtgatccagtcctgaggggc

SEQ ID NO. 184 IGLVl-101 (P)

>IGLV1-101* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccggcctccgtgtctggggccttgggccagagggtcaccatc tcctgcactggaagcagctccaatgttggttatagcagctatgtgggcttgtaccagcag ctcccaggaacaggcctcaaaaccatcatctataataccaatactcgaccctctggggtt cctgatcaattctctggctccaaatcaggcagcacagccacctgaccattgctggacttc aggctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc

SEQ ID NO. 185 IGLV1-103 (F)

>IGLV1-103* 01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactcagccaccctctgtgtctgcagccctggggcagagggtcaccatc tcctgcactggaagtaacaccaacatcggcagtggttatgatgtacaatggtaccagcag ctcccaggaaagtcccctaaaactatcatttatggtaatagcaatcgaccctcgggggtc ccggttcgattctctggctccaagtcaggcagcacagccaccctgaccatcactgggatc caggctgaggatgaggctgattattactgccagtcctatgatgacaacctcgatggtca

SEQ ID NO. 186 IGLV1-104 (P)

>IGLV1-104* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccagcttcagtgtctgggtccctgggccagaggatcaccatc tcctgcactaaaagcagctccaacatcggtaggtattatgtgagctgacaacagctccca ggaacaggccccagaaccgtcatctatgataataataactgaccctcgggggtccctgat caattttctggctctaaatcaggcagcacagccaccctgaccatctctaggctccaggct gaggacgatgctgattattactgctcgccatatgccagcagtctcagtgctgg

SEQ ID NO. 187 IGLV1-106 (F)

>IGLV1-106* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgttgactcaaccggcctcagtgtctgggtccctgggccagagggtcatcatc tcctgcactggaagcagctccagcattggcagaggttatgtgggctggtaccaacagctc ccaggaacaggccccagaaccctcatctatggtattagtaacctacccccgggagtcccc aatagattctctggttcgaggtcaggcagcacagccaccctgaccatcgctgagctccag gctgaggacgaggctgattattactgctcatcgtgggacagaagtctcagtgctcc

SEQ ID NO. 188 IGLV1-107 (P)

> IGLV1-107*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgctgactcagcccgccctcagtgtctgcggccttgggacagagggtcaccat ctcctgcactggaagcagcaccaacatcagcagtggttacgttgtacaatggtaccagca gctcccaggaaagtcccctaaaacaatctatggtactagcaagtgacccttggggatccc ggttcaattctctggctccaagtcaggcagcacagccaccctgaccatcactggtatcta ggctgaggacgaggctgattattactgccaatcctatgatgacaacctcgatggtca

SEQ ID NO. 189 IGLVl-110 (P)

> IGLV1-110*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtacggaatcaaccgccctcagagtctgcagccctgggacagagagtcaccatc tcctgcacgggaagcagatccaacattggcagtggttatgctgtacaatggtaccaacgg ctcacaggaaagtctccttaaaactatcatctatggtaatagcaatcaaccctcgggggt cctggatcaattctctggctccaagtgaggcagcacagccaccctgaccatcactgggat ccagtctgaggacgaggctgattattactgccagtcctatgatagaagtctctgtgctca SEQ ID NO. 190 IGLVl-111 (ORF)

>IGLV1-111* 01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggcctgagggtcaccatc tgctgcactggaagcagctccaacatcagtagttattatgtgggctggtaccaaccactc gcgggaacaggccccagaactgtcatctatgataatagtaaccgtccctcgggggtccct gatcaattctctggctccaagtcaggcagcacagccaccctgaccatctctcggctccag gctgaggacgaggctgattattacggctcatcatatgacagcagtctcaatgctgg

SEQ ID NO. 191 IGLV1-112 (P)

>IGLV1-112* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccagcctcagtgtctcagtccctgggtcagagggtcaccatc tcctgtactggaagcagctccaatgttggttataacagttatgtgagctggtaccagcag ctcccaggaacagtccccagaaccatcatctattataccaatactcgaccctatggggtt cctgatogattctctggctccaaatcaggcaactcagccaccctgaccattgctggaetc caggctgaggacgaggctgattattattgctcaacatatgacagcagtctcagtggtgc

SEQ ID NO. 192 IGLV1-113 (P)

>IGLV1-113-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgaatcagacgccctcagtgtcggggtccctgggccagagagtcgccatc tcctgctctggaagcacaaacatcagtaggtttggtgcgagctggtaacaacagctcctg ggaaaggcttcaaaactcctcctagacagtgatggggatcaaccatcagtggtccctgac tgattttccggctccaagtctggcaactcaggtgccctgaccatcactgggctccaggct gaggacgaggctgattattactgccagtcctttgatcccacacttggtgctca

SEQ ID NO. 193 IGLV1-114 (P)

>IGLV1-114* 01 I Canis lupus familiaris boxer | P | V-REGION | caggctttgctgactcagccaccctcagtgtctgaggccctgggacagagggtcaccatc tcctgcactggaagcagcaccaacatcggcagtggttatgatgtacaatggtaccagcag ctcccaggaaagtcccctcaaactatcgtatacggtaatagcaattgaccctcgggggtc ccagatcaattctctggctccaagtctcacaattcagccaccctgaccatcactgggctc cagactgaggacgaggctgattattactgccagtcctctgatgacaacctcga

SEQ ID NO. 194 IGLV1-115 (P)

>IGLV1-115* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccagcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagatatagtgtaggctgataccagcagctc ccgggaacaggccccagaactgtcatctatggtagtagtagccgaccctcgggggtcccc gatcgattctctggctccaagtcaggcagcacagccaccctgaccatctcagggctccag gctgaggacgaggctgattattactgttcaacatacgacagcagtctcaaagctcc

SEQ ID NO. 195 IGLV1-116 (F)

>IGLV1-116* 01 I Canis lupus familiaris boxer | F | V-REGION | cagcctgtgctcactcagccgccctcagtgtctgggttcctgggacagagggtcactatc tcctgcactggaagcagctccaacatccttggtaattctgtgaactggtaccagcagctc acaggaagaggccccagaaccgtcatctattatgataacaaccgaccctctggggtccct gatcaattctctggctccaagtcaggcaactcagccaccctgaccatctctgggctccag gctgaggacgagactgattattactgctcaacgtgggacagcaggctcagagctcc

SEQ ID NO. 196 IGLV1-118 (P)

GLVl-118* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactgaaagcagctccaacatcggtggatattatgtgggctggtaccaacagctc ccaggaacaggccccagaaccatcatctatagtagtagtaaccgaccctcaggggtccct gattgattctctggctccaggtcaggcagcacagccaccctgaccatctctgggctccag gctgaggacgaggctgattattactgctctacatgggacagcagtctcaaagctcc

SEQ ID NO. 197 IGLV1-118-2 (P)

>IGLV1-118-2* 01 I Canis lupus familiaris boxer | P | V-REGION | ctgcctgtgctgacccagccgccctcaaggtctgggggtctggttcagaggttcaccatc ttctgttctggaagcacaaacaacataggtgataattattttaactggtacaaacagctt ccaggaacggcccctaaaaccatcatctaagtggatcatatcagaccctcaggggtcctg gagagattctctgtctccaattctggcagctcagccaacctgaccatctctgggctccag gctgaggactaggctgattattattgctcatcctgggatgatagtctcaatgctcc

SEQ ID NO. 198 IGLV1-122 (P)

>IGLV1-122* 01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgctgactcagctgccctcagtgtctgcagccctgggacagagggtcaccatc tgcactggaagcagcaccaacatcggcagtggttattatacactatggtaccagtagctg caggaaagtcccctaaaactatcatctatggtaatagcaatcgacccttgagggtcccgg atcgattctctggctccaagtatggcaattcagccacgctgaccatcactgggctccagg ctgaggacgaggatgattattactgccagtcctctgatgacaacctcgatggtca

SEQ ID NO. 199 IGLV1-123 (P)

>IGLV1-123* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggtcagagggtcaccatc tcctgcactggaagcagctccaacatcggtgaatattatgtgagttggctccagcagctc ccgggaacacgccccagaaccgtcatctatagtagtagtaaccgaccctcaggggtccct gatcgattctctggctccaagtcaggtagcatagccaccctatctctgggctccaggctg aagacgaggctgattattactgtactacgtgggacagcagtctcaatgctgg

SEQ ID NO. 200 IGLV1-125 (F)

>IGLV1-125* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtccgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgggctggtaccaacagctc ccgggaacaggccccagaaccctcatctatggtaatagtaaccgaccctcaggggtcccc gatcggttctctggctccaggtcaggcagcacagccaccctgacoatctctgggctccag gctgaggatgaggctgattattactgctcatcgtgggacagcagtctcagtgctct

SEQ ID NO. 201 IGLV1-127 (P)

>IGLV1-127* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagcctccctcagtgtctgggtccctgggccagaggtcaccgtct cctgcactggaagctgcttcaacattggtagatatagtgtgagctggctccagcagctcc cgggaacaggccccagaaccatcatctattatgatcgtagccgaccctcaggggttcccg atcgattctctggctccaagtcaggcagcacagccaccctgaccatctctgggctccagg ctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaaggtca

SEQ ID NO. 202 IGLV1-129 (P)

>IGLV1-129* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccagtctcagtgtctggggccctgtgccagagggtcaccatc tcctgcactggaagcagctccaacattggttatagcagctgtgtgagctgatatcagcag ctcccaggaacaggccccagaaccatcatctatagtatgaatactctaccctctggggtt cctgatogattgtctggctccaggtcaggcaactcagccaccctaaccatctctgggetc caggctgaggacaaggctgactattactgctcaacatatgacagcagtctcaatgctca

SEQ ID NO. 203 IGLV1-130 (P)

>IGLV1-130* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgacccagctggcctcagtgtctgggtccctgggccagagggtcaccatc acctgcactggaagcagctccaacattggtagtgattatgtgggctggttccaacagctc ccaggaacaggccctagaaccctcatctaaggcaatagtaaccgaccctcgggggtccct gatcaattctctggctccaagtctggcagtacagccaccctgaccatctctgggctccag gctgaggatgatgctgattattactgcacatcatgggatagcagtctcaaggctcc

SEQ ID NO. 204 IGLV1-132 (ORF)

>IGLV1-132* 01 I Canis lupus familiaris boxer | ORF | V-REGION | cagtctgtgctgactcagcctccctcagtgtctgggaccctggggcaaagggtcatcatc tcctgcactggaatccccagcaacataaatttagaagaattgggaatcgctactaaggtg aactggtaccaacagctcccaggaaaggcacccagtctcctcatctatgatgatgatagc agaggttctgggattcctgatcgattctctggctccaagtctggcaactcaggcaccctg accatcactgggctccaggctgaggatgaggctgattattattgccaatcctatgatgaa agccttggtgtt SEQ ID NO. 205 IGLV1-133 (P)

>IGLV1-133* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagcctccctcagtgttcaggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacgtcggtagaggttatgtgatctggtaccaaagctcc tgggaacacgcccaagaaccctcatatatggtagtagtaaccaaccctcaggggtcccca atcgattctctggctccaggtcaggcagcacagacactctgacaatctctgtgttccagg ctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 206 IGLV1-135 (F)

>IGLV1-135* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgaatcagctgccttcagtgttaggatccctgggccagagaatcaccatc tcctgctctggaagcacgaatgacatcggtatgcttggtgtgaactggtaccaagagctc ccaggaaaggcccctaaactcctcgtagatggtactgggaatcgaccctcaggggtccct gaccgattttctggctccaaatctggcaactcaggcactctgaccatcactgggctccag gctgaggacgaggctgattattattgtcagtccactgatctcacgcttggtgctcc

SEQ ID NO. 207 IGLV1-136 (F)

>IGLV1-136* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgggctggtaccagcagctc ccaggaacaggccccagaaccctcatctatgatagtagtagccgaccctcgggggtccct gatcgattctctggctccaggtcaggcagcacagcaaccctgacoatctctgggctccag gctgaggacgaggctgattattactgctcagcatatgacagcagtctcagtggtgg

SEQ ID NO. 208 IGLV1-138 (F)

>IGLV1-138* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaatgttggttatggcaattatgtgggctggtaccagcag ctcccaggaacaagccccagaaccctcatctatgatagtagtagccgaccctcgggggtc cctgatcgattctctggctccaggtcaggcagcacagcaaccctgaccatctctgggctc caggctgaggatgaagccgattattactgctcatcctatgacagcagtctcagtggtgg

SEQ ID NO. 209 IGLV1-139 (F)

>IGLV1-139* 01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactccgctgccctcagtgtctgcggccctgggacagacggtcaccatc tcttgtactggaaatagcacccaaatcagcagtggttatgctgtacaatggtaccagcag ctcccaggaaagtcccctgaaactatcatctatggtgatagcaatcgaccctcgggggtc ccagatogattctctggcttcagctctggcaattcagccacactggccatcactgggetc caggatgaggacgaggctgattattactgccagtccttagatgacaacctcaatggtca

SEQ ID NO. 210 IGLV1-140 (P)

>IGLV1-140* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccggcctccgtgtctggggacttgggccagagggtcaccatc tcctgcactggaagcagctccaattttggttatagcagctatgtgggcttgtaccagcag ctcccaggaacaggccccagaaccatcatctataataccaatactcgaccctctggggtt cctgatcgattctctggctccaaatcaggcagcacagccacctgaccattgctggacttc aagctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc

SEQ ID NO. 211 IGLV1-140-1 (P)

>IGLV1-140-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtactgactcagccgccattagtgcttggggccctggccagagggtcaccttct cctgccttggaagagtcccagtattggtgattatggtgtgaaatggtacaagcagctcaa aaggacagaccccagacttctcatctatggcaatagcaattgatcctcgggtccccaatc aattttctggctctggttttggcatcactggctccttgaccacctatgggctccagactg aaaaataggctgattactagtgcttctccggtgatccag

SEQ ID NO. 212 IGLV1-141 (F)

>IGLV1-141* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccgacctcagtgtcggggtcccttggccagagggtcaccatc tcctgctctggaagcacgaacaacatcggtattgttggtgcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtgtacagtgttggggatcgaccgtcaggggtccct gaccggttttccggctccaactctggcaactcagccaccctgaccatcactgggcttcag gctgaggacgaggctgattattactgccagtcctttgataccacgcttggtgctca

SEQ ID NO. 213 IGLV1-143 (P)

>IGLV1-143* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccagtctcagtgtctggggccctgtgccagagggtcaccatc tcctgcactggaagcagctccaacattggttatagcagctgtgtgagctgatatcagcag ctcccaggaacaggccccagaaccatcatctatagtatgaatactctaccctctggggtt cctgatcgattgtctggctccaggtcaggcaactcagccaccctaaccatctctgggctc caggctgaggacaaggctgactattactgctcaacatatgacagcagtctcaatgctca

SEQ ID NO. 214 IGLV1-144 (F)

> IGLV1-144*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagcctccctcagtgttcaggtccctgggccagagggtcaccatc tcctgcactggaagcagctgcaacgtcggtagaggttatgtgatctggtaccaacagctc ctgggaacacgcccaagaaccctcatatatggtagtagtaaccaaccctcaggggtcccc aatcgattctctggctccaggtcaggcagcacagccactctgacaatctctgggttccag gctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 215 IGLV1-146 (P)

> IGLV1-146*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgaatcagctgccttcagtgttaggatccctgggccagagaatcaccatc tcctgctctggaagcacgaatgacatcggtatgcttggtgtgaactggtaccaagagctc ccaggaaaggcccctaaactcctcgtagatggtactgggaatcgaccctcaggggtccct gactgattttctggctccaaatctggcaactcaggcactctgaccatcactgggctccag gctgaggacgaggctgattattattgtcagtccactgatctcacgcttggtgctcc

SEQ ID NO. 216 IGLV1-147 (F)

>IGLV1-147* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgggctggtaccagcagctc ccaggaacaggccccagaaccctcatctatgataatagtaaccgaccctcgggggtccct gatcgattctctggctccaagtcaggcagcacagccaccctgacoatctctgggctccag gctgaggacgaggctgattattactgctcaacatacgacagcagtctcagtggtgg

SEQ ID NO. 217 IGLV1-149 (F)

>IGLV1-149* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaatgttggttatggcaattatgtgggctggtaccagcag ctcccaggaacaggccccagaaccctcatctatcgtagtagtagccgaccctcgggggtc cctgatcgattctctggctccaggtcaggcagcacagcaaccctgaccatctctgggctc caggctgaggatgaagccgattattactgctcatcctatgacagcagtctcagtggtgg

SEQ ID NO. 218 IGLV1-150 (F)

>IGLV1-150* 01 I Canis lupus familiaris boxer | F | V-REGION | caggctgtgctgactccgctgccctcagtgtctgcggccctgggacagacggtcaccatc tcttgtactggaaatagcacccaaatcggcagtggttatgctgtacaatggtaccagcag ctcccaggaaagtcccctgaaactatcatctatggtgatagcaatcgaccctcgggggtc ccagatogattctctggcttcagctctggcaattcagccacactggccatcactgggetc caggatgaggacgaggctgattattactgccagtccttagatgacaacctcgatggtca

SEQ ID NO. 219 IGLV1-151 (F)

> IGLV1-151*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgcgctgactcaaacggcctccatgtctgggtctctgggccagagggtcaccgtc tcctgcactggaagcagttccaacgttggttatagaagttatgtgggctggtaccagcag ctcccaggaacaggccccagaaccatcatctataataccaatactcgaccctctggggtt cctgatcgattctctggctccatatcaggcagcacagccaccctgactattgctggactc caggctgaggacgaggctgattattactgctcatcctatgacagcagtctcaaagctcc SEQ ID NO. 220 IGLV1-151-1 (P)

>IGLV1-151-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcagccactgttagggcctgggttcctggccagagggtcaccctct cctgccctggaagagtctcagttttggtgattatggtgtgaaacggtacaggaagctcgc atggacagaccccagactcctcatctatggcaatagcaattgattctcgggtccccagtc tattttctggctctggttttggcatcactggctccttgaccacctccgggctccagactg aaaaataggctgatttctagtgcttc

SEQ ID NO. 221 IGLV1-152 (P)

>IGLV1-152* 01 I Canis lupus familiaris boxer | P | V-REGION | caatctgtgctgatccagccggcctcagtgtcgggatccctgggccagagagtcaccatc tcctgctctggaaggacaaacaacatcggtaggtttggtgcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtggacagtgatggggattgaccgtcaggggtccct gaccggttttccggctccaggtctggcagctcagccaccctgaccatcactggggtccag gctgaggatgaggctgattattactgccagtcctttgatcccacgcttggtgctca

SEQ ID NO. 222 IGLV1-154 (P)

>IGLV1-154* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccgtcctcagtgtccgggtccctgggccagagggtcactgtc ccctgcactggaagcagctccaacattggtagatatagtgtgagctggctatatctgctg gctccagcagctcccgggaacaggccccagaaccatcatctattatgattgtagccgacc ctcaggggttcccgatcgattctctggctccaagtcaggcagcacagccaccctgaccat ctctgggctccaggctgaggacgaggctgattattactgctcatcctatgacagcagtct caaaggtca

SEQ ID NO. 223 IGLV1-155 (F)

>IGLV1-155* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagcctccctcagtgtccgggttcctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgcactggtaccaacagctc ccaggaacaggccccagaaccctcatctatggtattagtaaccgaccctcaggggtcccc gatcgattctctggctccaggtcaggcagcacagccactctgacaatctctgggctccag gctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 224 IGLV1-157 (F)

>IGLV1-157* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccggcctcagtgtctgggtccctgggccagagggtcaccatc tcctgcactggaagcagctccaacatcggtagaggttatgtgggctggtaccagcagctc ccaggaacaggccccagaaccctcatctatgataatagtaaccgaccctcgggggtccct gatcgattctctggctccaagtcaggcagcacagccaccctgacoatctctgggctccag gctgaggacgaggctgattattactgctcaacatacgacagcagtctcagtggtgg

SEQ ID NO. 225 IGLV1-158 (F)

>IGLV1-158* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgaatcagctgccttcagtgttaggatccctgggccagagaatcaccatc tcctgctctggaagcacgaatgacatcggtatgcttggtgtgaactggtaccaagagctc ccaggaaaggcccctaaactcctcgtagatggtactgggaatcgaccctcaggggtccct gaccgattttctggctccaaatctggcaactcaggcactctgaccatcactgggctccag gctgaggacgaggctgattattattgtcagtccactgatctcacgcttggtgctcc

SEQ ID NO. 226 IGLV1-159 (F)

>IGLV1-159* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagcctccctcagtgttcaggtccctgggccagagggtcaccatc tcctgcactggaagcagctgcaacgtcggtagaggttatgtgatctggtaccaacagctc ctgggaacacgcccaagaaccctcatatatggtagtagtaaccaaccctcaggggtcccc aatcgattctctggctccaggtcaggcagcacagccactctgacaatctctgggttccag gctgaggatgaggctgattattactgctcatcctgggacagcagtctcagtgctct

SEQ ID NO. 227 IGLV1-160 (P)

>IGLV1-160* 01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgtgctgactcaaccagtctcagtgtctggggccctgtgccagagggtcaccatc tcctgcactggaagcagctccaacattggttatagcagctgtgtgagctgatatcagcag ctcccaggaacaggccccagaaccatcatctatagtatgaatactctaccctctggggtt cctgatcgattgtctggctccaggtcaggcaactcagccaccctaaccatctctgggctc caggctgaggacaaggctgactattactgctcaacatatgacagcagtctcaatgctca

SEQ ID NO. 228 IGLV1-161 (P)

>IGLV1-161-1* 01 | Canis lupus familiaris boxer | P | V-REGION | caaggtcagctgccctgaggacagagtccatgacaggtcagggcagaaacagggactctg aatccagctctgagtcaggacacatcaggagtgtccaatatgtgtcctgctaccaacagc tccatgagtgggcagtcaaatcctcatgtattatgatggcttgaccttctgtggaccctg gtccattctctgcctccatgtctggcagctctggctctctggccattgctgggctgagcc aggaggatgaggtcatgcttcactgcccctccagtgacagcatttcaaggat

SEQ ID NO. 229 IGLV1-162 (F)

> IGLV1-162*01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgtgctgactcagccgacctcagtgtcggggtcccttggccagagggtcaccatc tcctgctctggaagcacgaacaacatcggtattgttggtgcgagctggtaccaacagctc ccaggaaaggcccctaaactcctcgtgtacagtgatggggatcgaccgtcaggggtccct gaccggttttccggctccaactctggcaactcagacaccctgaccatcactgggcttcag gctgaggacgaggctgattattactgccagtcctttgataccacgcttgatgctca

SEQ ID NO. 230 IGLV2-31 (F)

> IGLV2-31* 01 I Canis lupus familiaris boxer | F | V-REGION | cagtctgccctgactcaaccttcctcggtgtctgggactttgggccagactgtcaccatc tcctgtgatggaagcagcagtaacattggcagtagtaattatatcgaatggtaccaacag ttcccaggcacctcccccaaactcctgatttactataccaataatcggccatcagggatc cctgctcgcttctctggctccaagtctgggaacacggcctccttgaccatctctgggctc caggctgaagatgaggctgattattactgcagcgcatatactggtagtaatactttc

SEQ ID NO. 231 IGLV2-31-1 (P)

>IGLV2-31-1*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctaacctaattgagcccccctttttgtccaggattctaggatggactgtcactgtc tcctgtgttttaagcagctgtgacatcaggagtgataatgaaatatcctggtaccaatag cacccgagcatgactcagaaattcctgatttactataccagttcttgggcatcagatatc cctgattgctttcctggctcccagtctggaaacatggcctgtctgaccatttccaggctc caggctaatgatgacgctgattatcattgttacttatatgatggtagtggcgctttt

SEQ ID NO. 232 IGLV2-32 (P)

>IGLV2-32*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgccctgactcagcctccctcgatgtctgggacactgggacagacoatcatcatt tcctgtactggaagcggcagtgacattgggaggtatagttatgtctcctggtaccaagag ctcccaagcacgtcccccacactcctgatttatggtaccaataatcggccattagagatc cctgctcgcttctctggctccaagtctggaaacacagcccccatgaccatctctgggctt caggctgaagatgaggctaattattactgttgctcatatacaaccagtggcacaca

SEQ ID NO. 233 IGLV2-32-1 (P)

>IGLV2-32-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagtctgccttgacccaacctccctttgtgtctgggactttgagacaaactgtcacatct cttgcaatggaagcagcagccacactggaacttataaccctacctctggcaccagcaatg tctggaaaggcccccacactccagatagatgctgtgagttctttgccttcagggcttcca gctctgtcctcaggctctgagtctagcaacacagcctccagtccatttttggactgcacc ctgaggacaaggctgattattactgattgtccagggacagccagag

SEQ ID NO. 234 IGLV3-1 (P)

>IGLV3-1* 01 I Canis lupus familiaris boxer | P | V-REGION | gccaacaagctgactcaatccctgtttatgtcagtggccctgggacagatggccaggatc acctgtgggagagacaactctggaagaaaaagtgctcactggtaccagcagaagccaagc caggctcccgtgatgcttatcgatgatgattgcttccagccctcaggattctctgagcaa ttctcaggcactaactcggggaacacagccaccctgaccattagtgggcccccagcgagg acgcggctattactgtgccaccagccatggcagttggagcacct SEQ ID NO. 235 IGLV3-1-1 (P)

>IGLV3-1-1* 01 I Canis lupus familiaris boxer | P | V-REGION | tccaatgtactgacacagccacccttggtgtcagtgaacctgggacagaaggccagcctc acctgtggaagaaacagcattgaagataaatatgtttcatggtcccagcaggagccaggc caggcccccatgctggtcatctattatagtacacaagaaaccctgagcgattttctgcct ccagctctagctcggggtacatgatcaccctgaccaacagtggggcctaggacaaggacg aggatggctattactgtcagtcctatgacagtagtggtactcct

SEQ ID NO. 236 IGLV3-2 (F)

>IGLV3-2* 01 I Canis lupus familiaris boxer | F | V-REGION | tectatgtgctgactcagtcaccctcagtgtcagtgaccctgggacagacggccagcatc acctgtaggggaaacagcattggaaggaaagatgttcattggtaccagcagaagccgggc caagcccccctgctgattatctataatgataacagccagccctcagggatccctgagcga ttctctgggaccaactcagggagcacggccaccctgaccatcagtgaggcccaaaccaac gatgaggctgactattactgccaggtgtgggaaagtagcgctgatgct

SEQ ID NO. 237 IGLV3-3 (F)

>IGLV3-3* 01 I Canis lupus familiaris boxer | F | V-REGION | tcctatgtgctgacacagctgccatccaaaaatgtgaccctgaagcagccggcccacatc acctgtgggggagacaacattggaagtaaaagtgttcactggtaccagcagaagctgggc caggcccctgtactgattatctattatgatagcagcaggccgacagggatccctgagcga ttctccggcgccaactcggggaacacggccaccctgaccatcagcggggccctggccgag gacgaggctgactattactgccaggtgtgggacagcagtgctaaggct

SEQ ID NO. 238 IGLV3-4 (F)

>IGLV3-4* 01 I Canis lupus familiaris boxer | F | V-REGION | tccactgggttgaatcaggctccctccatgttggtggccctgggacagatggaaacaatc acctgctccggagatatcttagggaaaagatatgcatattggtaccagcataagccaagc caagcccctgtgctcctaatcaataaaaataatgagcgggcttctgggatccctcactgg ttctctggttccaactcgggcaacatggccaccctgaccatcagtggggcccgggctgag gacgaggctgactattactgccagtcctatgacagcagtggaaatgct

SEQ ID NO. 239 IGLV3-7 (P)

>IGLV3-7* 01 I Canis lupus familiaris boxer | P | V-REGION | tcctatgtgctgactctgctgctatcagtgaccgtgaacctgggacagaccaccagcatc acctgtggtggagacagcattggagggagaactgtttactggtaccagcagaagcctggc cagcgccccctgctgattatctataatgatagcaattgaccctcagggatccctgcctga ttctctggctccaactcagggaacagggcctccctaaccatcattggggcctgggcctaa gacgagtctgagtattacggagaggtgtgggacagcagtgctaaggct

SEQ ID NO. 240 IGLV3-7-1 (P)

> IGLV3-7-l*01 I Canis lupus familiaris boxer | P | V-REGION | tcctatatgctgactcagcagccattggcaagtgtaaacctcagccagtgggccagcacc acctgtggtggagataacattggagaaaaaaccgtccaatggaaccagcagaagcctggc taagctcccattacggctatctataaaggtagtgatctgccctcagggatccctgagcaa ttccctggccccaatttggggaacggggcctccctgaacatcagcggggctaagccgacg acgaggctattactgccagtcagcagacattagtggtaaggct

SEQ ID NO. 241 IGLV3-8 (F)

>IGLV3- 8* 01 I Canis lupus familiaris boxer | F | V-REGION | tectatgtgctgacacagctgccatccgtgagtgtgaccctgaggcagacggcccgcatc acctgtgggggagacagcattggaagtaaaagtgtttactggtaccagcagaagctgggc caggcccctgtactgattatctatagagatagcaacaggccgacagggatccctgagcga ttctctggcgccaactcggggaacacggccaccctgaccatcagcggggccctggccgag gacgaggctgactattactgccaggtgtgggacagcagtactaaggct

SEQ ID NO. 242 IGLV3-9 (P)

>IGLV3- 9* 01 I Canis lupus familiaris boxer | P | V-REGION | tccactgggttgaatcaggctccctccgtgttgctggcactgggacagatggcaacaatc acctgatccagagatgtctttgggaaaaatatgcatattggtaccagcagaagccaagcc aagcccctgtgctcctaatcaataaaaataatgagcaggattctgggatccctgaccggt tctctggctccaactcgggcaacacggccaccctgaccatcagtggggcccgggccgagg acgaggctgactattactgccagtcctatgacagcagtggaaatgtt

SEQ ID NO. 243 IGLV3-11 (F)

>IGLV3-11*01 I Canis lupus familiaris boxer | F | V-REGION | tectatgtgctgtctcagccgccatcagcgactgtgactctgaggcagacggcccgcctc acctgtgggggagacagcattggaagtaaaagtgttgaatggtaccagcagaagccgggc cagccccccgtgctcattatctatggtgatagcagcaggccgtcagggatccctgagcga ttctccggcgccaactcggggaacacggccaccctgaccatcagcggggccctggccgag gacgaggctgactattactgccaggtgtgggacagcagtactaaggct

SEQ ID NO. 244 IGLV3-13 (P)

>IGLV3-13*01 I Canis lupus familiaris boxer | P | V-REGION | tectatgtactgactcagctgccatcagtgactgtgaacctgggacagaccaccagcatc acctgtggtggagacagcattggagggagaactgtttactggtaccagcagaagcctggc cagcgccccctgctgattatctataatgatagcaattggccctcagagatccctgcctga ttctctggctccaactcagggaacagggcctccctaaccatcattggggcctgggcctaa gatgagtctgagtattacggagaggtgtgggacagcagtgctaaggct

SEQ ID NO. 245 IGLV3-13-1 (P)

>IGLV3-13-1*01 I Canis lupus familiaris boxer | P | V-REGION | tcctatatgctgactcagcagccattggcaagtgtaaacctcagccagtgggccagcacc acctgtggtggagataacattggagagaaaactgtccaatggaaccagcagaagcctggc taagctctcattatggctatctataaaggtagtgatctaccctcagggatccctgagcaa ttccctggccccaactcgggtcggggcctccctgaacatcagcggggctacgccgacgac taggctattactgccagtcagcagacattagtggtaaggct

SEQ ID NO. 246 IGLV3-14 (F)

>IGLV3-14*01 I Canis lupus familiaris boxer | F | V-REGION | tcctatgtgctgacacagctgccatccatgagtgtgaccctgaggcagacggcccgcatc acctgtgagggagacagcattggaagtaaaagagtttactggtaccagcagaagctgggc caggtccctgtactgattatctatgatgatagcagcaggccgtcagggatccctgagcga ttctccggcgccaactcggggaacacagccaccctgaccatcagcggggccctggccgag gacgaggctgactattactgccaggtgtgggacagcagtactaaggct

SEQ ID NO. 247 IGLV3-15 (P)

>IGLV3-15*01 I Canis lupus familiaris boxer | P | V-REGION | tccactgggttgaatcaggctccctccgtgttggtggccctgggacagatggaaacaatc acctgctcgagagatgtcttagggaaaagatatgcatataggtaccagcataagccaagc caagcccctgtgctcctaatcaataaaaataatgagcaggattctgggatccctgaccgg ttctctggctccaactcgggcaacacggccaccctgaccatcagtggggcccgggctgag gacgaggctgagtattactgccagtcctatgacagcagtggaaatgtt

SEQ ID NO. 248 IGLV3-18 (P)

>IGLV3-18*01 I Canis lupus familiaris boxer | P | V-REGION | tcctatgtgctgacacagctgccatccgtgaatgtgacccagaggcagacggcccgcatc acctgtgggggagacagcattggaagtaaaagtgtttactggtaccagcagaagctgggc caggcccctgttgattatctatagagacagcaacaggccgacagggatccctgagcgatt ctctggcgccaacacggggaacatggccaccctgactatcagcggggccctggccgtgga cgaggctgactattactgccaggtgtgggacagcagtgctaaggct

SEQ ID NO. 249 IGLV3-19 (ORF)

>IGLV3-19*01 I Canis lupus familiaris boxer | ORF | V-REGION | tcccctgggctgaatcagcctccctccgtgttggtggccctgggacagatggcaacaaac acctgctccggagatgtcttagggaaaagatatgcatattggtaccagcataagccaagc caagcccctgtgctcctaatcaataaaaataatgagctgggttctgggatccctgaccga ttctctggctccaactcgggcaacacggccaccctgaccatcagtggggcccgggccgag gacgaggctgactattactgccagtcctatgacagcagtggaaatgct SEQ ID NO. 250 IGLV3-21 (F)

>IGLV3-21*01 I Canis lupus familiaris boxer | F | V-REGION | tectatgagctgactcagccaccatccgtgaatgtgaccctgagggagacggcccacatc acctgtgggggagacagcattggaagtaaatatgttcaatggatccagcagaatccaggc caggcccccgtggtgattatctataaagatagcaacaggccgacagggatccctgagcga ttctctggcgccaactcagggaacacggctaccctgaccatcagtggggccctggccgaa gacgaggctgactattactgccaggtgggggacagtggtactaaggct

SEQ ID NO. 251 IGLV3-23 (P)

>IGLV3-23*01 I Canis lupus familiaris boxer | P | V-REGION | tectatgtactgactcagctgccatcagtgactgtgaacctgggacagaccaccagcatc acctgtggtggagacagcattggagggagaactgtttactggtaccagcagaagcctggc cagcgccccctgctgattatctataatgatagcaattggccctcagagatccctgcctga ttctctggctccaactcagggaacagggcctccctaaccatcattggggcctgggcctaa gacgagtctgagtattacggagaggtgtgggacagcagtgctaaggct

SEQ ID NO. 252 IGLV3-23-1 (P)

>IGLV3-23-l*01 I Canis lupus familiaris boxer | P | V-REGION | tcctatatgctgactcagcagccattggcaagtgtaaacctcagccagtgggccagcacc acctgtggtggagataacattggagaaaaaactgtccaatggaaccagcagaagcctggc taagctcccattacggctatctataaaggtagtgatctgccctcagggattcctgagcaa ttccctggccccaactcgggaaacggggcctccctgaacatcagcggggctaagccgacg actaggctattactgccagtcagcagacattagtggtaaggct

SEQ ID NO. 253 IGLV3-24 (F)

>IGLV3-24*01 I Canis lupus familiaris boxer | F | V-REGION | tectatgtgctgacacagctgccatccgtgagtgtgaccctgaggcagacggcccgcatc acctgtgggggagacagcattggaagtaaaaatgtttactggtaccagcagaagctgggc caggcccctgtactgattatctatgatgatagcagcaggccgtcagggatccctgagcga ttctccggcgccaactcggggaacacggccaccctgaccatcagcggggccctggccgag gatgaggctgactattactgccaggtgtgggacagcagtactaagcct

SEQ ID NO. 254 IGLV3-25 (ORF)

>IGLV3-25*01 I Canis lupus familiaris boxer | ORF | V-REGION | tccactgggttgaatcaggcttcctccgtgttggtggccctgggacagatggaaacaatc acctgctcgagagatgtcttagggaaaagatatgcatataggtaccagcataagccaagc caagcccctgtgctcctaatcaataaaaataatgagcaggattctgggatccctgaccgg ttctctggctccaactcgggcaacacggccaccctgaccatcagtggggcccgggctgag gacgaggctgagtattactgccagtcctatgacagcagtggaaatgtt

SEQ ID NO. 255 IGLV3-26 (F)

>IGLV3-26*01 I Canis lupus familiaris boxer | F | V-REGION | tcctatgtgctgacacagctgccatccgtgaatgtgaccctgaggcagccggcccacatc acctgtgggggagacagcattggaagtaaaagtgttcactggtaccaacagaagctgggc caggcccctgtactgattatctatggtgatagcaacaggccgtcagggatccctgagcga ttctctggtgacaactcggggaacacggccaccctgaccatcagtggggccctggccgag gacgaggcttactattactgccaggtgtgggacagcagtgctcaggct

SEQ ID NO. 256 IGLV3-27 (F)

>IGLV3-27*01 I Canis lupus familiaris boxer | F | V-REGION | tccagtgtgctgactcagcctccttcagtatcagtgtctctgggacagacagcaaccatc tcctgctctggagagagtctgagtaaatattatgcacaatggttccagcagaaggcaggc caagtccctgtgttggtcatatataaggacactgagcggccctctgggatccctgaccga ttctccggctccagttcagggaacacacacaccctgaccatcagcggggctcgggccgag gacgaggctgactattactgcgagtcagaagtcagtactggtactgct

SEQ ID NO. 257 IGLV3-28 (F)

>IGLV3-28*01 I Canis lupus familiaris boxer | F | V-REGION | tectatgtgttgactcagctgccttcagtgtcagtgaacctgggaaagacagccagcatc acctgtgagggaaataacataggagataaatatgcttattggtaccagcagaagcctggc caggcccccgtgctgattatttatgaggatagcaagcggccctcagggatccctgagcga ttctctggctccaactcggggaacacggccaccctgaccatcagcggggccagggccgag gatgaggctgactattactgtcaggtgtgggacaacagtgctaaggct

SEQ ID NO. 258 IGLV3-29 (F)

>IGLV3-29*01 I Canis lupus familiaris boxer | F | V-REGION | tccagtgtgctgactcagcctccctcggtgtcagtgtccctgggacagacggcgaccatc acctgctctggagagagtctgagcagatactatgcacaatggtatcagcagaagccaggc caagcccccatgacagtcatatatggggacagagagcgaccctcagggatccctgaccga ttctccagctccagttcagagaacacacacaccttgacaatcagtggagcccaggctgag gatgaggctgaatattactgtgagatatgggacgccagtgctgatgat

SEQ ID NO. 259 IGLV3-30 (F)

>IGLV3-30*01 I Canis lupus familiaris boxer | F | V-REGION | tcctacgtggtgacccagccaccctcagtgtcagtgaacctgggacagacggccagcatc acctgtgggggagacaacattgcaagcacatatgtttcctggcagcagcagaagtcgggt caagcccctgtgacgattatctatcgtgatagcaaccggccctcagggatccctgagcga ttctctggctccaactcggggaacacggccaccctgaccatcagcagggcccaggccgag gatgaggctgactattactgccaggtgtggaagagtggtaataaggct

SEQ ID NO. 260 IGLV4-5 (F)

>IGLV4-5* 01 I Canis lupus familiaris boxer | F | V-REGION | ttgcccgtgctgacccagcctacaaatgcatctgcctccctggaagagtcggtcaagctg acctgcactttgagcagtgagcacagcaattacattgttcagtggtatcaacaacaacca gggaaggcccctcggtatctgatgtatgtcaggagtgatggaagctacaaaaggggggac gggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatctcc aacatcaagtctgaagatgaggatgactattattactgtggtgcagactatacaatcagt ggccaatacggttaagc

SEQ ID NO. 261 IGLV4-6 (P)

>IGLV4-6* 01 I Canis lupus familiaris boxer | P | V-REGION | ttgcccgtgctgacccagcctccaagtgcatctgcctccctggaagcctcggtcaagctc acatgcactctgagcagtgagcacagcagttactatatttactggtatgaacaacaacaa ccagggaaggcccctcggtatctgatgagggttaacagtgatggaagccacagcaggggg gacgggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatc tccaacatccagtctgaggatgaggcagattattactgtggtgcacccgctggtagcagt age

SEQ ID NO. 262 IGLV4-10 (F)

>IGLV4-10*01 I Canis lupus familiaris boxer | F | V-REGION | ttgcccgtgctgacccagcctacaaatgcatctgcctccctggaagagtcggtcaagctg acctgcactttgagcagtgagcacagcaattacattgttcattggtatcaacaacaacca gggaaggcccctcggtatctgatgtatgtcaggagtgatggaagctacaaaaggggggac gggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatctcc aacatcaagtctgaagatgaggatgactattattactgtggtgcagactatacaatcagt ggccaatacggttaagc

SEQ ID NO. 263 IGLV4-12 (P)

>IGLV4-12*01 I Canis lupus familiaris boxer | P | V-REGION | ttgcccgtgctgacccagcctccaagtgcatctgcctccctggaagcctcggtcaagctc acatgcactctgagcagtgagcacagcagttactatatttactggtatcaacaacaacca gggaaggcccctcggtatctgatgaaggttaacagtgatggaagccacagcaggggggac gggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatctcc aacatccagtctgaggatgaggcaggttattactatggtgtacccctggtagcagtagc

SEQ ID NO. 264 IGLV4-16 (ORF)

>IGLV4-16*01 I Canis lupus familiaris boxer | ORF | V-REGION | ttgcccatgctgacccagcctacaaatgcatctgcctccctggaagagtcggtcaagctc acatgcactttgagcagtgagcacagcaattacattgttcaatggtatcaacaacaacca gggaaggcccctcggtatctgatgcatgtcaggagtgatggaagctacaacaggggggac gggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatctcc aacatcaagtctgaagatgaggatgactattattacagtggtgcatactatacaatcagt ggccaatacggttaagc

SEQ ID NO. 265 IGLV4-17 (P)

>IGLV4-17*01 I Canis lupus familiaris boxer | P | V-REGION | ttgcccatgctgacccagcctccaagtgcatctgcctccctggaagcctcggtcaagctc acatgcactctgagcagtgagcaaagcagttactatatttactggtatcaacaacaacaa ccagggaaggcccctcggtatctgatgaaggttaacagtgatggaagccacagcagggcg tcgggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatct ccaacatccagtctgaggatgaggcagattattactgtggtgtacccactggtagcagta gc

SEQ ID NO. 266 IGLV4-20 (ORF)

>IGLV4-20*01 I Canis lupus familiaris boxer | ORF | V-REGION | ttgcccatgctgaccgagcctacaaatgcatctgcctccctggaagagtcagtcaagctc acctgcactttgagcagtgagcacagcaattacattgttcgatggtatcaacaacaacca gggaaggcccctcggtatctgatgtatgtcaggagtgatggaagctacaacaggggggac gggatccccagtcgcttttcaggctccagctctggggctgaccgctatttaaccatctcc aacatcaagtctgaagatgaggctgagtattattacggtggtgcagactataaaatcagt gaccaatatggttaaga

SEQ ID NO. 267 IGLV4-22 (F)

>IGLV4-22*01 I Canis lupus familiaris boxer | F | V-REGION | ttgcccgtgctgacccagcctccaagtgcatctgcctgcctggaaacctcggtcaagctc acatgcactctgagcagtgagcacagcagttactatatttactggtatcaacaacaacaa ccagggaaggcccctcggtatctgatgaaggttaacagtgatggaagccacagcaggggg gacgggatccccagtcgcttctcaggctccagctctggggctgaccgctatttaaccatc tccaacatccagtctgaagatgaggcagattattactgtggtgtacccgctggtagcagt age

SEQ ID NO. 268 IGLV5-34 (P)

>IGLV5-34*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgctgacccagccgccctccctctctgcatccctgggatcaacagccagactc acctgcaccctgagcagtggcttcagtgttggcagctactacafatactggtaccagtag aagccagggagccctccccggtatctcctgtactaactactactcaagtacacagctggg ccccggggtccccagccatttctctggatccaaagacaactcggccaatgcagggctcct gctcacctctgggctgcagcctgaggacgaggctgactactactgtgctacaggttattg ggatgggagcaactatgcttacc

SEQ ID NO. 269 IGLV5-38 (P)

>IGLV5-38*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgcatccctgggaacagcggccagaaat acctgcactctgagcagtgacctcagtgttggcagctgtgctataagctgatcccagcag aagccagggagccctccctggtatctcctgaactactaaacacacccatgcaagcaccag gactcacatctgtagccgcttctctggatttgaggatgcctctgccagtgcagggctctg ctcatctctggaggctgaccatcactgtgctaagatcatggcagtgggggcagctagtgt taca

SEQ ID NO. 270 IGLV5-38-1 (P)

>IGLV5-38-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccgtcctctctgcatccctgggaacaacagccagactca cctgcaccctgagcagtggcttcaatatgtggggctaccatatattctggtaccagcaga agccagggagccctccccggtatctgctgaacttctactcagataagcaccagggctcca aggacacctcggccaatgcagggatcctgctcatctctgggctccagcctgaggacgagg ctgactactactgtaaaatctggtacagtggtctggt

SEQ ID NO. 271 IGLV5-40-1 (P)

>IGLV5-40-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctctgctacccagccacccccttctctgcgtctccaggtactacagccagacccac ctgcaccctgagcagtggcaacagtgttggcagctgttccttataacggctcccacaaag acagagggccctccctggtatctgctgaggttcccctctaatagacaccatgtctctgga tccacacataccttggccaatgcagggctcctgctcat SEQ ID NO. 272 IGLV5-42 (P)

>IGLV5-42*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgaccaagtgccctctctttctgcatctcctggaacaacagtcagactca cttgcacctggagcagtggctccagcactggcagctactatatacactggttccagagcc acagagccagagccacagagctctccctggtatctcctgtactactactcagactcagat aagcaccagggctctggggttctcagctctgtctcctgatccaaggatgcctcagttatt ggagggctctctcatctctgggctgcagcctgaggattagactgaccttcactgtctaat cagaaacaataatgcttct

SEQ ID NO. 273 IGLV5-47 (P)

>IGLV5-47*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagctgccctccctctctgcataccggggaacaaactccagatgt acctacaccctgagcagtgtcgccaactactaaacatacttctcaaagagaatacagggc accttccacagtacatcctgtactactactcagactcaagtgoatgattgggatttgggg tcccaggcacttctctggatccaaagatgcctcagccaatgcagggatcctgctgatctc tgggctgcagccagaggacaagtctgactgtcactgtgctacagatcatggcagtgggag cagcttccgatact

SEQ ID NO. 274 IGLV5-47-1 (P)

>IGLV5-47-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctgacccagccacactccctctctgcatatcagggagaaacagccacacat acctgcaccctgagcggtggcttcagtgttggcagctgccatatatactggatccagaag aagccagagagccctccctgatgtctcctgaactactactaagactcagataaggcctcg acgtccccagccctactctgaatccaaagacaccttgcccaaggtgggaatcctgctcat ctctgggctgcagccggaggacaaggctgtctcttactgtataatatggcacagtggttc tggtcacagggaca

SEQ ID NO. 275 IGLV5-48-1 (P)

>IGLV5-48-l*01 I Canis lupus familiaris boxer | P | V-REGION | caccctgtgctgacccagctgccctccctctctgcatccctgggaacaacagccagactc atgtgcaccctgagcagtggctgcagtggtggccatacgctggttccagcagccaggagg cctcctgagtacctgctgatggtctactgagactcaccagggccccggtggccccagccg cttctctggctccaaggacacctcggccaatgcagggctcctgctcatctctaggctgca gcctgaggacgaggctgactgtcactgtgttacagaccatggcagtgggagcagctcccg aaactca

SEQ ID NO. 276 IGLV5-49-1 (P)

>IGLV5-49-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctggcccagcttccccccacctccctctgcatctccaggaacaacagccag actcacatgaaccatgagcagtggcttcatcgttggcgctgctacatatactggttccaa cagaagccagggagcaccgccccagtatctcctgaggttctactcagactcagataagca ctagggctcaacgaccccagccctgttctggatctgaagacacctccgccgaagcagggc ctctgctcatctctgggctgcagcgtgaggacaaggctgactcttatgggacaatctggc acagtggtcctggtcacagggacaca

SEQ ID NO. 277 IGLV5-51 (P)

>IGLV5-51*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagctgccctccctttctgcatccctgggaacaacagccagactc acatgcaccctgagcagcggctgcagcggtggccacacattggttccagcagccaggagg cctcctgagtacctgctgatggtctactgagactcaccagggccccggtgttgccagcct cttctctggctccaaggacacctcggccaatgcaggactcctgctcatctctgggctgca gcctgaggatgaggctgactgtcactgtgctacagaccatggcagtgggagcagctccgg atact

SEQ ID NO. 278 IGLV5-53 (P)

>IGLV5-53*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagctgccctccctttctgcatccctgagaacaacagccagactc acctgcaccctgagcagtggctgcagtggtggccatatgctggttccagcagccaggaag cctcctgagtatctgctgacggtcttctgagactcaccagggccccgaggtccccagcct cttctctggctccaaggacacctcagccaatgcaggactcctgctcatctctgggctgca gcctgaggatgaggctgactgtcactgtgctacagaccatggcagtgggagcagctcccg atact

SEQ ID NO. 279 IGLV5-53-1 (P)

>IGLV5-53-l*01 I Canis lupus familiaris boxer | P | V-REGION | caccctgggctgacccagtcgtcctccctctctgcatccctgggaacaacagccagactc acctgcaccctgagcagtggcttcagaaatgacaggtatgtaataagttggttccagcag aaatcagggagcccttcctggtgtctcctgtattattactcgaactcaagtacacatttg ggctctgaggttcccagctgcttctctggatccaagacaaggccacacccacactgagta gacccctctctgggtgggtctagagctccagctccacctgaggctgatgcacaattgcag

SEQ ID NO. 280 IGLV5-57-1 (P)

>IGLV5-57-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctggcccagctgccctccctctctgcatctccaggaacaacagccagactc acatgaaccatgagcagtggcttcattgttggtggctgctacatatactggttccaacag aagccagggagcatgccccccagtatctcctgaggttctactcagactcagataagcacc aggtctcaacatccccagcccggctctggatctgaagacactcagccgaagcagggcctc tgctcatctctgggctgcagcatgaggacaaggctgactcttactgtacaatctggcaca gtggtcctggtcacagggaca

SEQ ID NO. 281 IGLV5-58-1 (P)

>IGLV5-58-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccattgccctccctctctgcatcctgggaaataacaaccagactca cctgcactctgagcagcggctgcagcggtggccatacagtggttccagcagcaaggaagc ctcctgagtacctgctgacgttetactgagactcaccagggctctagggtccccagccac ttctctggtttcaaggacaccacggccaatgcagggcact

SEQ ID NO. 282 IGLV5-59 (P)

>IGLV5-59*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagtcgccctccctctcggcatctttggaacaacagtcagactca cctgtaccctgatcagtggctccagtgttggcagctattacatcaactggttccagaaga agccacggagccctccccagtatctcctgtactactacttagactcagataagcaccagg gctctggggtccccagctgcttctcctgatccaaggatgcctcagtcattggaggacacc ctcatctctgaactgcagcctgaggactagactgaccttcgctgtctaatcagaaacaat aatgettet

SEQ ID NO. 283 IGLV5-62 (P)

>IGLV5-62*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagcctccctctctctctgcatctctgggaacaatagccagacaa acatgcagcctgagcaggggctacagtatggggacttatgtcatacgctggttccagcag tagcaagaaactctcctgagtatctgctgaggttatactgagcctcagcaggtctctggg gaccccagctgagtctttagatccaagatgcctcagccaattcagggctcctgcttatct ctgtgctgcagcctgaggacaagggttactattactgttctgtacatcatggaattgtga gcagctatacttacc

SEQ ID NO. 284 IGLV5-64 (F)

>IGLV5-64*01 I Canis lupus familiaris boxer | F | V-REGION | cagcttgtggtgacccagccgccctccctctctgoatccctgggatcatccgccagaetc acctgcaccctgagcagtggcttcagtgttggcagttattctgtaacttggttccagcag aagccagggagccctctctggtacctcctgtactaccactcagactcagataagcaccag ggctccagggtccccagccgcttctctggatccaaggacacctcggccaatgcagggctc ctgctcatctctgggctgcagcctgaggatgaggctgactactactgtgcctccgctcat ggcagtgggagcaactaccattact SEQ ID NO. 285 IGLV5-67-1 (P)

>IGLV5-67-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagccagtgctgacccagctgccctccttctctgtatctctgggaacaacagtcagactc acctgcaccctgagcagtgttggcagctactaaacatccttttcaaggagaaaccaagga gccccccaccccggtatctcctatactactattcagactcagataaaccccaggtctctg gggtccccagccacttctctgcatccaaagactcctaggccaatgcagggctcctgctcg cctctgggctgcagcctgaggacgaggctgactatcactgtgctataaatcatgacagtg ggagtagttcctgatact

SEQ ID NO. 286 IGLV5-70-1 (P)

>IGLV5-70-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttggtgacccagcgccctccctctctgcatctcctgaaacaacagtcagactca catgcaccctgagcagtggccccagtgctggcagctactacatacactggttccagtgga agccacggtgcccgccccggtatctcctgtactactactcagactcagatgagcaccagg gctctggggtccccagccgcttctcctgatccaaggatgcctcagccagggcagggctcc ctcatctctgggctacagtctgaggtctacactgaccttcactgtctaatcggaaacaat aatgtttct

SEQ ID NO. 287 IGLV5-72-1 (P)

>IGLV5-72-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagcgacctccctctctgcatccctgggaacaacagccagactca cctgcaccctgagcagcggctgaagcggtggccatacgctggttccagcagccaggaagc ctcctgagtacctgctgatggtctactgagactcaccaggctatggggtccccagcatct tctctggctccaaggacacctcggccaatgcagggctcctgctcatctctgggctgcagc ctgaggtcgaggctgactgtcactgtgctacagaccatggcagtgggagcagctcccgat act

SEQ ID NO. 288 IGLV5-76 (P)

>IGLV5-76*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagtcgccctccctctcagcatctttggaacaacagtcagactca cctgtaccctgatcagtggctccagtgttggcagctattacatcaactggttccagaaga agccacggagccctccccagtatctcctatactactacttagactcagataagcaccagg gctctggggtccccagctgcttctcctgatccaaggatgcctcagtcattggagggcacc ctcatctctgagctgcagcctgaggactagactgaccttcgctgtctaatcggaaacaat aatgcttct

SEQ ID NO. 289 IGLV5-77 (P)

>IGLV5-77*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccaccctccctctctgcatccccgggaacaacagccagactc acctgcaccctgagcagtggcttcagtgttggtgactatgacatgtactggtaccagaag aagccaggaagcccccaccccgggatctcctgtactactactcagactcatataaacacc agggctccggggtctccagcagcttctctggatccaaggatacctcagccaatacagggc tcctgctcatctctgggccacagcctgaggacgaggctgactactactgtgctacagatc atggcagtgagagcaggtactcttacc

SEQ ID NO. 290 IGLV5-77-1 (P)

>IGLV5-77-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctctgctacccagcacccccttcgctgcgtttccaggtactacagccagaatcacc tgcaccctgagcaggggcatcagtgttgggagctgttccttataacggctcccgcagagg cagggagccctgcctggtatctgctgaggttcccctctaatagacaccacatctctggat ccaaagaaacctcggccaatgcagggctcctgctcattgttgtgctgccacctgacaact agtctatcagtggtggttgaggactaggactattactgggatgctttggttt

SEQ ID NO. 291 IGLV5-78-1 (P)

>IGLV5-78-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttgctgatccagcgccctccctctctgcatctcctggaacaacagtcagactca cctgcacccagagcagtggcccctgtgttggcagctactacatacactggttccagtgga agccatggagccctccctggtatcttctgtactactaatcagactcagatgagcaccagg gctctggggtccccagccgcttctcctgatccaaggatgcctcagccagagcagggctcc ctcatctctggactgcagcctgaggactagactgaccttcactgtctaatcagaaacaat aatgttt SEQ ID NO. 292 IGLV5-83-1 (P)

>IGLV5-83-l*01 I Canis lupus familiaris boxer | P | V-REGION | tgcaggtccctgtcccagcctttgccctccctctttgcatctcctggaagaacagtcaga tccacctgcacccagagcagtggcccctgtgttggcagctactacatacaccggttccag tggaagccacggagccgtctccatatctcctgtactactactcagactcagatgagcacc agagctctggagtccccaactgcttctcctgatccaaggatgcctcagggaaggcagggc tccctcatctctgggctacaggctgaggacaagactgacctttactgtctaatccaaaac aataatgtttct

SEQ ID NO. 293 IGLV5-85 (F)

>IGLV5-85*01 I Canis lupus familiaris boxer | F | V-REGION | cagcctgtgctgacccagccaccctccctctctgcatccctgggatcaacagccagaccc acctgcaccctgagcagtggcttcagtgttggaagctaccatatactctggttccagcag aagtcagagagccctccccggtatctcctgaggttctactcagattctaatgaacaccag ggtcccggggtccccagccgcttctctggatccaaggacacctcaacctatgcagggctc ttgctcatctctgggctgcagcctgaggacgaggctgactactactgtgctacagaccat ggcagtgggagcagctacacttacc

SEQ ID NO. 294 IGLV5-86-1 (P)

>IGLV5-86-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttgctgacccagcgccctccctctctgcatctcctggaacaaaagtcagactca cctgcatccagagcagtggatccagcgttggcagctactacatacactggttccagtaga agccatggagccctccccagtatctcctgtactactacttagactcagataagcactagg cctatggggaacccagatccttcccctgatccaaggatgcctcagtcaatgcagggtcaa agagaggggattatttagagtggacaattggggcctttggccaggag

SEQ ID NO. 295 IGLV5-88-1 (P)

>IGLV5-88-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagccagtgcagacccagctgccctccttctctgtacctctgggaacaacagccagactc acctgcaccctgagcagtgttggcggccagtaaacatccttttcaaggagaaaccaagga gccccccagtctctcctgtactattacccagactcagataaaccccaggtctctggggtc cccagccacttctctgaatccaaagactcctaggccaatgcagggctcctgctegoctet gggctgcagcctgaggacgaggctgactatcactgtgctgtaaatcatgacagtgggagc agctccggatact

SEQ ID NO. 296 IGLV5-89-1 (P)

>IGLV5-89-l*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtggtgacccagcttccttctctgcatccctgggaacaacagccagactcacat gcaccctgagctgtggcttcagtattgatagatatgctataaactggttccagcagaagg cagagagccttccctggtacctactgtgctattactggtactcaagtacacagttgggct tcagcgtccccagctgcatctctggatccaagacaaggccacattcacaaacgagtagac ccatctctggttgggtctagagctccagccccacctgagactgatgcacaattgcagc

SEQ ID NO. 297 IGLV5-92-2 (P)

>IGLV5-92-2*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtatagacccagtcaccctccctttctgcatctttggaacaacagtcagagtca cctgtaccctgagcagtggctccagtgttggcagctactacatatactggttccaggaga agccatggagcaatccccggtatctcctgtactactcaggctcagatgagcaccagggct ctgggatccgtagctgcttctcctgatacaatgatgcctcagccaaggcagagctcccta atctctgggctgcagcctgaggactatactgaccttcactgtctaatcagaaacaataat cctttt

SEQ ID NO. 298 IGLV5-94-1 (P)

>IGLV5-94-l*01 I Canis lupus familiaris boxer | P | V-REGION | tagcctgtgctgacccagcgccctcccactctgcatccctgggaacaacagccagactca cctgcgccctgagcagcggctgcagcagtgaccatacgctggttccagcagccagaaggc ctcctgagtacctgctgacggtetactgagactcaccagcgccccggggtcctcagcctc ttctctggctccaaggacacctcggccaatgcagggcactcagatgg SEQ ID NO. 299 IGLV5-95 (P)

>IGLV5-95*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgatgacccagctgtcctccctctctgcatccctggaaacaacaaccagacac acctgcaccctgagcagtggcttcagaaataacagctgtgtaataagttgattccagcag aagtcagggagccctccctggtgtctcctgtactattactcagactcaagtatacatttg ggctctgaggttcccagctgcttctctggatccaagacaaggccacacccacactgagta gacccatccctgggtgggtctagagctccagccccactggaggctgatgcacaattgcag c

SEQ ID NO. 300 IGLV5-96-1 (P)

>IGLV5-96-l*01 I Canis lupus familiaris boxer | P | V-REGION | caacctttgcggacccagcgcactccctctgcatctcctggaacaacagttagactcatc tgcacccagagcagtggccccagtgttggcagctactacaaacactggttccagcagaag ccacggagccctccccggtacttcctgtactacttctcagactcagatgagcaccagggc tctggggaccgcagccacttctcctgatccaaggatgactcaggaaaggcagggctccct catctctgggctacagcctgaggactagactgaccttcactgtctaatcagaaacaataa tgcttct

SEQ ID NO. 301 IGLV5-97-1 (P)

>IGLV5-97-l*01 I Canis lupus familiaris boxer | P | V-REGION | ttaaaaccaaccaaaccaaaccaaaccaaaacaaaacaaaacaaaataacagccagattc acctgctccctgagcagtggcttcagtgttggtggctataacacactggtaccagcagaa gccagggagccctccctgttacctcctgtactactactcagaatcagataaacaccatgg ctccgggatcaccagctgcttccctggccctatggacacctcggccaatgcagggctcct gctcatctcagggctgcagcctgaggacgaggctgactactactgcggtatactccacag cagtgggagcagctactcttacc

SEQ ID NO. 302 IGLV5-97-2 (P)

>IGLV5-97-2*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgaggacacactcctccttcctctctgcacctttgggatcatcaaccagactc acctgcatccttcccagggcctgaatgttggcaggtactgaacatactggacaaggagaa tcaaggagacatcaggagttccctcagatccagataagtgccagggcacggggttctcag ccacttctatggatctaatgatgcctcaggcaatgcaggtctcctgctcatgtctgggct gcagcctgaggacgaggctgactatgactatgctgcacattgtggggtgggagcagctcc cgatact

SEQ ID NO. 303 IGLV5-97-3 (P)

>IGLV5-97-3*01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgoatccctgggaacaacagccagaetc acctgcaccctgagcagcagctgcagcggtggccatatgctggttccagcatgcaagagg cctcctgagtacctgctgatggtctactgagactcaccagggccctggggtccccagcct cttctctggctccaaggaagcctcggccaatgcagggctcctgctcatctctgggctgca gcctgagaatgaggctgactgtcactgtgctacagaccatggcagtgggaacagctccca atact

SEQ ID NO. 304 IGLV5-101-1 (P)

>IGLV5-101-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttgctgacccagcgtcctccctctctgcatctcctggaacaacagtcagactca catgtaccctgagcagtggccccggtgctggcagctactacacacactggttccagcaga ggccacagagtcctccccggtatctcctgtactactactcagactcagatgatctccagg gctccgggttccccagccactcctcctgatccaaggatgcctcagccagggcagggctcc catctctggggtacagcctgaggactacactgaccttcactgtctaatcggaaacaataa tgtttct SEQ ID NO. 305 IGLV5-103-1 (P)

>IGLV5-103-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctggcccagctgccccccacctccctctgcatctccaggaacaacagccag actcacatgaaccatgagcagtggcttcattgttggcagctgctacatatactggttcca acagaagccagggagcccccctcccccaatatctcttgaggttgtattcagaatcagata aacaccagggctcaatgtccccagccctgctctggatctgaagacacctccgccgaagca gggcctctgctcatctctgggctgcagcgtgaggacaaggctgactcttactgtacaatc tgg

SEQ ID NO. 306 IGLV5-105 (P)

>IGLV5-105* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgoatccctgggaacaacagccagaetc acctgcaccatgagcagcagctacagtggtggccatacactggttccagcagccaggagg cctcctgagtacctgctgatggtctactgagatttaccagggccccggggtccccagccg cttctctggctccaaggacatctcggccaatgcagggctcctgctcatctctgggctgta gcctgaggacgaggctgactgtcactgtgctacagaacatggcagcgggagcagctccca atact

SEQ ID NO. 307 IGLV5-105-1 (P)

>IGLV5-105-1* 01 I Canis lupus familiaris boxer | P | V-REGION | ctgcctctgctacccagccaccgccttctctgcatctccaggtactacagccagacccac ctgcaccctgaacagtggcatcagtattcgcagctgttccttataatggctcccgcaaag gcagggagccctgcctggtatctgctaaggttgtactctaataaataccatggctctagg gtcccaagccacatctctggatccaaagaaacctc

SEQ ID NO. 308 IGLV5-106-1 (P)

>IGLV5-106-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttgctgacccagcgtcctccctctctgcatctcctggaacaacagtcagactca cctgtatccagagcagtggccccagtgttggcagctactacatacaccggttccagcgga aaccacggagccctcccctgtatctcctgtactactactcagactcagataagcactagg cctacagggtccccagctgcttctcctgatccatggatgcctcagccagtgcagtgctcc ctcatctctgggctacagcctgaggactagactgaccttcactgtctaatcggaaacaat aatgettet

SEQ ID NO. 309 IGLV5-109 (F)

>IGLV5-109* 01 I Canis lupus familiaris boxer | F | V-REGION | cagcttgtgctgacccagccgccctccctctctgcatccctgggatcaacaaccagactc acctgcaccctgagcagtggcttcagtgttggtggctatagcatatactggcaccagcag aagccagggagcactccctggtacctcctgtactactactcaagtacagagttgggacct ggggtccccagctgcttctctggatccaaagacacctcagccaatgtagggctcctgctc atctcagggctgcagcctgaggatgagactgactactactgtgctataggtcacggcagt gggagcagctacacttacc

SEQ ID NO. 310 IGLV5-110-1 (P)

>IGLV5-110-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctggcccagctgcccccccacctccctctgcatctccaggaataacagcca gactcacatgaaccatgagcagtggcttcattgttggccgctgctacatatactgattcc aacagaagccaaggagcccccgctccaccagtatctcctgatattctactcagactcaga taagcaccagggctcaacgtccccagccctgctctgaatctgaagacacctccgcgaagc agggcttctgctcatctctgggctcagcgtgaggacaaggctgactcttactgtacaatc tgg

SEQ ID NO. 311 IGLV5-111-1 (P)

>IGLV5-111-1* 01 I Canis lupus familiaris boxer | P | V-REGION | tagcctgtgctgacccagtgctctccctctctgcatccctgggaacaacagccagactcc cctgcaccctgagcagcggctgcagcggtgtccatacgcaggttccagcagccaggaggc ctcctgaatacctgctgatggtctacggtgactcaccagggccccggggtccccagccgc ttctctggctccgaggacacctcggccaatgcagggctcctgctcatctctgggctgcag cctgaggacaagactgactgtcactgtgctacagaccatggcagtaggagcagttcccaa tact SEQ ID NO. 312 IGLV5-111-2 (P)

>IGLV5-lll-2* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagctgcccttcctctctgcatccctggagacaacaagcagatgt acctacacccagagcggtgtcggcagctactacacatactcatcaaggacaatccaggga gacctccctggtatttcctgtactactactcagactcaactacatggttgggatttggtg tccccaaccacttctctgtatccaaagatgcctcagccaatgcagggctcctgctcatct ctgggctgcagccagaggacaaggatgactgtcactgtgctgcattcagatcatggcagt gggagcagctcccgatact

SEQ ID NO. 313 IGLV5-113-2 (P)

>IGLV5-113-2* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctttgctgatccagtgccctccctctctgcatctcctggaacaagagtcagactca cctgcacccagagcagtggccccagggttggcagctactacatacactggttgcagcgga aaccacggagccctcctcagtatctcctgtactactactcagaatcagatgagcaccagg gctctggggtccccagccacttctcctgatccaaggatgcctcaggcaaggcagggctcc ctcatccctgggctacagcctgagggctagactgaccttcactgtctaatccgaaacaat aatgtttct

SEQ ID NO. 314 IGLV5-114-1 (P)

>IGLV5-114-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagccagggctggcccagctgccctccctctctgcatctccaggaacaacagccagactc acatgaaccatgaacagtggcttcattcttggcggctgatacatatacttgttccaacag aaaccagggaacccccgctccccgtattgcctgaggttctactcagactcagataagcac cagggctcaacatccccagccctgctctggatctgaagacacctcaactgaagcagggcc tctgctcatctctggatgtccagcgtgaggacaaggttgattcttactgtacaatctggc acagtggtcctggt

SEQ ID NO. 315 IGLV5-115-1 (P)

>IGLV5-115-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctctgctgacccagccaccctccctctctgcatccctgggaacaagacccagagtc acctgcaccctgagcaacaactgcagtggtggccatacgctggttccagcagccaggaag cctcctgaatacctattgatggtttactgagacttaccagggcccccggggccccagctg cttctctggctccaaggacaccttggccaatgcaggactcctgctcatctctgggctgta gcctgaggatgaggctgactgtcactgtgctacagaccatggcagtgggagcagctcccg atact

SEQ ID NO. 316 IGLV5-118-1 (P)

>IGLV5-118-1* 01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtggtgacccagcttccttctctgcatccctgggaacaacagccagattcacat gcaccctgagctatggcttcagtattgatagatatgttataagctggttccagcagaagg cagagagccttccctggtacctactgtactattactgatactcaagtacacagttgggct tcggcattcccagctgcgtctctggatccaagacaaggccacattcacaaatgagtagac ccatctctggttgggtctagagctccagccccacctgagactgatgcacaattgcagcca cattgtcttgatatcggaaa

SEQ ID NO. 317 IGLV5-124-1 (P)

>IGLV5-124-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtatagacccagtcaccctccctttctgcatctttggaacaacagtcagactca cctgtaccctgagcagtggctccagtgttggcagctactacatatactggttccaggaga agccatggagcaatccccggtatctcctgtactattcaggctcagatgagcaccagggct ctgggatccctagctgcttctcctgatccaaggatgcctcagccaaggcagagctccctc atctctgggctgcagcctgaggactagactgaccttcactgtctaatcagaaacaataat gcttct

SEQ ID NO. 318 IGLV5-125-1 (P)

>IGLV5-125-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagcgccctcccactctgcatccctgggaacaacagccagactca cctgcaccctgagcagcggctgcagcggtggccatatgctggttccagcagccagaaggc ctcctgagtacctgctgacggtetactgagactcaccagggcccctgggtcctcageetc ttctctgactccaaagacacctcggccaatgcagggcactcagatggctgtgaagttcat acaacagggtcctcatgggggctcatggtaccacttcacgttt SEQ ID NO. 319 IGLV5-126 (P)

>IGLV5-126* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgatgacccagctgtcctccctctcagcatccctggaaacaacaacaagactc acctgaaccctgagcagtggcttcagaaatgacagatgtgtaataagttggttccagcag aagtcagggagccctccctggtgtctcctgtactattactcggactcaagtacacatttg ggctctgaggttcccagctgcttctctggatccaagacaaggccacacccacactgagta gacccatccccgggtgggtctagagctccagccccactggaggctgatgcacaattgcag c

SEQ ID NO. 320 IGLV5-128-1 (P)

>IGLV5-128-1* 01 I Canis lupus familiaris boxer | P | V-REGION | caacctttgcggacccagcgccctccctctctgcatctcctggaacaacagttagactca tctgcacccagagcagtggccccagtgttggcagctactacaaacactggttccagcaga agecacggagccctccccggtacctcctgtactactactcagactcagatgagcaccagg gctctggggaccacagccacttctcctgatccaaggatgcctcaggaaaggcagggctcc ctcatctctgggctacagcctgaggactagactgaccttcactgtctaatcagaaacaat aatgettet

SEQ ID NO. 321 IGLV5-129-1 (P)

>IGLV5-129-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgaccagctgccctctctgcatccctgggaacaacaggcagatgtactta caccctgagcagttttggcagctactacacatactegtcaaggagaatacagggagacct ccctggtatttcctgtactactactcagactcaactacatggttgggatttggggtcccc aaccacttctctggatccaaagatgcctcagccaatgcagggctcctgctcatctctggg ctgcagccagaggacaaggatgactgtcactgtgctgcatacatatcaaggcagtggaag cagctcccaatact

SEQ ID NO. 322 IGLV5-129-2 (P)

>IGLV5-129-2* 01 I Canis lupus familiaris boxer | P | V-REGION | ctgcctgtgctgacccagtgccctccctctctgcatccctgggaacaacagccagactca cctgcaccctgagcagtggctgcagcggtggccatatgctggttccagcagccaggaggc ctcctaagtacctgctgatggtctactgagactcatcacggtcctggggtccctagcctc ttctctggctccaaggacacctcggccaatgcagggctcctgctcatctctgggctgcag cctgaggacgaggctgactgtcattgtgctacagaccatggcagtgggagcagctcctga tact

SEQ ID NO. 323 IGLV5-131 (F)

>IGLV5-131* 01 I Canis lupus familiaris boxer | F | V-REGION | cagcctgtgctgacccagccaccctccctctctgcatccctgggaacaacagccagactc acctgcaccctgagcagtggcttcagtgttggtgactatgacatgtactggtaccagcag aagccagggagccctccccgggatctcctgtactactactcggactcatataaaaaccag ggctctggggtctccaaaagcttctctggatccaaggatacctcagccaatgcagggctc ctgctcatctctgggctgcagcctgaggacgaggctgactactactgtgctacagatcat ggcagtgagagcagctactcttacc

SEQ ID NO. 324 IGLV5-132-1 (P)

>IGLV5-132-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtatagacccagtcaccctccctttctgcatctttggaacaacagtcagactca cctgtaccctgagcagtggctccagtgttggcagctactacatatactggttccaggaga agccatggagcaatccccggtatctcctgtactactcaggctcagatgagcaccagggct ctgggatccctagctgtttctcctgatccaaggatgcctcagccaaggcagagctccctc atctctgggctgcagcctgaggactatactgaccttcactgtctaatcagaaacaataat gettet SEQ ID NO. 325 IGLV5-134 (P)

>IGLV5-134* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgcatccctgggaacaacagccagactc acctgcaccatgagcagcagctgcagcggtggccatatgctggtaccagcatgcaagagg cctcctgagtacctgctgatggtctactgagactcaccagggccctggggtccccagcct cttctctggctccaaggacaccttggccaatgcagggctcctgctcatctctgggctgca gcctgagaatgaggctgactgtcactgtgctacagaccatggcagtgggaacagctccca atact

SEQ ID NO. 326 IGLV5-134-1 (P)

>IGLV5-134-1* 01 I Canis lupus familiaris boxer | P | V-REGION | taaaaccaaaccaaaccaaaccaaaccaaaacaaaacaaaacaaaataacagccagattc acctgctccctgagcagtggcttcagtgttggtggctataacacactggtaccagcagaa gccagggagccctccctgttacctcctgtactactactcagaatcagataaacaccatgg ctccgggatcaccagctgcttccctggccctatggacacctcggccaatgcagggctcct gctcatccttgggctgcagcctgaggacgaggctgactactactgcggtatactccacag cagtgggagcagctactcttacc

SEQ ID NO. 327 IGLV5-135-1 (P)

>IGLV5-135-1* 01 I Canis lupus familiaris boxer | P | V-REGION | aagcctgtgctgacccagcgccctccctctctgcatccctgggaacaacagccagactca cctgcaccctgagcagcggctggagtggtggctataggctggttccagcagccaggaagc ctcctgagtacctgctgatggtctactgagactcaccaggctatggggtccccagcatct tctctggctccaaggaagcctcggccaatgcagggctcctgctcatctctggcctgcagc ctgaggtcgaggctgactgtcactgtgctacagaccatggcagtgggagcagctcccgat ac

SEQ ID NO. 328 IGLV5-137-1 (P)

>IGLV5-137-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctaacccagtcgctctccctcttgacatctttggaacaacagtcagactca cctgtaccgtgaacagtggctccagtgttggcagctattacatcaactggttccagtata agccatggagctctccctagtatcacctgtactactacttagactcagataagcaccagg gctctggggtccccagctgcttctcctgatccaaggatgcctcagtcattggagggcacc ctcatctctgggctgcagcctgaggactagactgaccttcacgtctaatcagaaacaata atgcttct

SEQ ID NO. 329 IGLV5-137-2 (P)

>IGLV5-137-2* 01 I Canis lupus familiaris boxer | P | V-REGION | ctgcctgtgctgacccagccgccctccctctctgoatccctgggatcaacagccagaetc acctgcacactgagcagtggctgcagcggtggccatatgctggttccagcagccaggagg cctcctgtgtacctgctgatggtctactgagactcaccagggccccagtgtccccagcca ctactctggtttcaaagacacctcggccaatgcaggtcactcagatagctgcgaaattca tacaacaagggtcctcatggggactcatgggcaccccttcagattttcctgcctgcatga acag

SEQ ID NO. 330 IGLV5-138-1 (P)

>IGLV5-138-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagggatggcccagctgttcccccacctccctctgcatctccaggaacaacagccagact cacatgaaccatgagcagtggcttcattgttggcggctgctacatatactggttccaaca gaagccagggagtccccttccccccatatctcctgagtttctactcagactcagataagc accagggctcaaaatccccagccctgttctggatctgaagacacctcagccaaagcagcg cctctgctcatctctgggctgcagggtgaggataagaatgactcttactctacaatctgg

SEQ ID NO. 331 IGLV5-139-1 (P)

>IGLV5-139-1* 01 I Canis lupus familiaris boxer | P | V-REGION | caacctttgcggacccagtgccctccctctctgcatctcctggaacaacagttagactca tctgcacccagagcagtggccccagtgttggcagctactacaaacactggttccagcaga agccacggagccctccccagtacctcctgtactacttctcagactcagatgagcaccagg gctctggggactgcagccacttcccctgatccaaggatgcctcaggaaagcagggctccc tcatctctgggctacagcctgaggactagactgaccttcactgtctaatcagaaacaata atgcttcttacagt SEQ ID NO. 332 IGLV5-145 (P)

>IGLV5-145* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgcatccctgggaacaacattcagactc acctgcaccctgagcagcagctgcagcggtggccatatgctggttccagcatgcaagagg cctcctgagtacctactgatggtctactgagactcaccagggccctggggtccccagcct cttctccggctccaaggacaccttggccaatgcagggctcctgctcatctctgggctgca gcctgagaatgaggctgactgtcactgtgctacagaccatggcagtgggaacagctccca atact

SEQ ID NO. 333 IGLV5-145-1 (P)

>IGLV5-145-1* 01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgacgacacactcctccttcctctctgcacctttgggatcatcaaccagactc acctgcatccttcccagggcctgaatgttggcaggtactgaacatactggacaaggagaa tcaaggaggcatcaggagttccctcagatccagataagtgccagggcacggggttctcag ccacttctatggatctaatgatgcctcaggcaatgcaggtctcctgctcatgtctgggct gcagcctgaggacgaggctgactatgactatgctgcacattgtggggtgggagcagctcc cgatact

SEQ ID NO. 334 IGLV5-146-1 (P)

>IGLV5-146-1* 01 I Canis lupus familiaris boxer | P | V-REGION | aagcctgtgctgacccagcgccctttctctctgcatccctgggaacaacagccagactca cctgcaccctgagcagcggctggagtggtggctataggctggttccagcagccaggaagc ctcctgagtacctgctgatggtctactgagactcaccaggctatggggtccccagcatat tctctggctccaaggaagcctcggccaatgcagggctcctgctcatctctgggctgcagc ctgaggtcgaggctgactgtcactgtgctacagaccatggcagtgggagcagctcccgat act

SEQ ID NO. 335 IGLV5-148 (P)

>IGLV5-148* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcaccaaggatccatcactctcagtgtttccaggagggacagtcacattc acatgtggcctcagctctgggtcagtctttacaagtaactaccccagctggtaccagcag acccatggccgggctcctcacatgcttatctacagcacaagcagctgcccccccggggtc cctgatcgcttctctggatccatctctgggaacaaagttgccctcaccatcacaggagcc cagcctgaggatgagactattattgttcactgcgtatgggtagtacattta

SEQ ID NO. 336 IGLV5-148-1 (P)

>IGLV5-148-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctaacccagtcgccctccctcttgacatctttggaacaacagtcagactca cctgtaccgtgaacagtggctccagtattggcagctattacatcaactggttccaggaga agccatggagctctccctggtatcacctatactacttcttagactcagataagcaccagg gctctggggtccccagctgcttctcctgatccaaggatgcctcagtcattggagggcacc ctcatctctgggctgcagcctgaggactagactgaccttcactgtctaatcagaaacaat aatgcttct

SEQ ID NO. 337 IGLV5-148-2 (P)

>IGLV5-148-2* 01 I Canis lupus familiaris boxer | P | V-REGION | ctgcctgtgctgacccagccgccctccctctctgcatccctgggatcaacagccagactc acctgcacactgagcagtggctgcagcggtagccatatgctggttccagcagccaggagg cctcctgggtacctgctgatggtctactgagactcaccagggccccagtgtccccagcca ctactctggatgcaaagacacctcggccaatgcaggt

SEQ ID NO. 338 IGLV5-149-1 (P)

>IGLV5-149-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagggatggcccagctgttcccccacctccctctgcatctccaggaacaacagccagact cacatgaaccatgagcagtggcttcattgttggcggctgctacatatactggttccaaca gaagccagggagtccccttccccccatatctcctgagtttctactcagactcagataagc accagggctcaaaatccccagccctgttctggatctgaagacacctcagccaaagcagcg cctctgctcatctctgggctgcagggtgaggataagaatgactcttactctacaatctgg SEQ ID NO. 339 IGLV5-150-2 (P)

>IGLV5-150-2* 01 I Canis lupus familiaris boxer | P | V-REGION | caacctttgcggacccagcgcactccctctgcatctcctggaacaacagttagactcatc tgcacccagagcagtggccccagtgttggcagctactacaaacactggttccagcagaag ccacggagccctccccggtacttcctgtactacttctcagactcagatgagcaccagggc tctggggaccgcagccacttctcctgatccaaggatgactcaggaaaggcagggctccct catctctgggctacagcctgaggactagactgaccttcactgtctaatcagaaacaataa tgcttct

SEQ ID NO. 340 IGLV5-154-1 (P)

>IGLV5-154-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgatgacccagctgtcctccctctctgcatccctggaaacaacaaccagacac acctgcaccctgagcagtggcttcagaaataacagctgtgtaataagttgattccagcag aagtcagggagccctccctggtgtctcctgtactattactcagactcaagtatacatttg ggctctgaggttcccagctgcttctctggatccaagacaaggccacacccacactgagta gacccatccctgggtgggtctagagctccagccccactggaggctgatgcacaattgcag c

SEQ ID NO. 341 IGLV5-155-1 (P)

>IGLV5-155-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctaacccagtcgctctccctcttgacatctttggaacaacagtcagactca cctgtaccgtgaacagtggctccagtgttggcagctattacatcaactggttccagtata agccatggagctctccctagtatcacctgtactactacttagactcagataagcaccagg gctctggggtccccagctgcttctcctgatccaaggatgcctcagtcattggagggcacc ctcatctcggggctgcagcctgaggactagactgaccttcactgtctaatcagaaacaat aatgcttctaacagtga

SEQ ID NO. 342 IGLV5-157-1 (P)

>IGLV5-157-1* 01 I Canis lupus familiaris boxer | P | V-REGION | | cccagcgccctttctctctgcatccctgggaacaacagccagactcacctgcaccctgag cagcggctagagtggtggctataggctggttccagcagccaggaagcctcctgagtacct gctgatggtctactgagactcaccaggctatggggtccccagcatcttctctggctccaa ggacacctcggccaatgcagggctcctgctcatctctgggctgcagcctgaggtcgaggc tgactgtcactgtgctacagaccatggcagtgggagcagctcccgata

SEQ ID NO. 343 IGLV5-158-1 (P)

>IGLV5-158-1* 01 I Canis lupus familiaris boxer | P | V-REGION | ataacagccagattcacctgctccctgagcagtggcttcagtgttggtggctataacaca ctggtaccagcagaagccagggagccctccctgttacctcctgtactactactcagaate agataaacaccatggctccgggatcaccagctgcttccctggccctatggacacctcggc caatgcagggctcctgetcatctcagggctgcagcctgaggacgaggctgactactactg cggtatactccacagcagtgggagcagctactcttacc

SEQ ID NO. 344 IGLV5-158-2 (P)

>IGLV5-158-2* 01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgacgacacactcctccttcctctctgcacctttgggatcatcaaccagactc acctgcatccttcccagggcctgaatgttggcaggtactgaacatactggacaaggagaa tcaaggaggcatcaggagttccctcagatccagataagtgccagggcacggggttctcag ccacttctatggatctaatgatgcctcaggcaatgcaggtttcctgctcatgtctgggct gcagcctgaggacgaggctgactatgactatgctgcacattgtggggtgggagcagctcc cgatact

SEQ ID NO. 345 IGLV5-158-3 (P)

>IGLV5-158-3* 01 I Canis lupus familiaris boxer | P | V-REGION | cagcctgtgctgacccagccgccctccctctctgcatccctgggaacaacattcagactc acctgcaccctgagcagcagctgcagcggtggccatatgctggttccagcatgcaagagg cctcctgagtacctactgatggtctactgagactcaccagggccctggggtccccagcct cttctctggctccaaggacaccttggccaatgcagggctcctgctcatctctgggctgca gcctgagaatgaggctgactgtcactgtgctacagaccatggcagtgggaacagctccca atact SEQ ID NO. 346 IGLV7-32-2 (P)

>IGLV7-32-2*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtggtgactccagagcccttctgaccatccccaggagtgacagtcacttttacc tgtgactccagcactggagagtcattaatagtgactatccacgttagttccagcagaagc ctagacaaactcgcaccacacacacaacaaacactcacggactcccacccagttctcagg ctccctccaggctcaaaactgccctcacctttttggggtcccagcctgagaaagaaggtg agtactaccatatgctggtctatcttggttcttgg

SEQ ID NO. 347 IGLV7-33 (P)

>IGLV7-33*01 I Canis lupus familiaris boxer | P | V-REGION | | caggctgtggtgactcaggaaccctcactgaccgtgtccctggagggacagtcactctca cctgtgcctccagcactggcgaggtcaccaatggacactatccatactggttccagcaga agcctggccaagtccccaggacattgatttataatacacacataatactcctggacccct acccggttctcaggctgcctctttgggggcaaagctgccttgaccatcacaggggcccag cccgaggatgaagctgaggactactgctggctagtatatatggtaatagg

SEQ ID NO. 348 IGLV7-36-1 (P)

>IGLV7-36-l*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtggtgattcaggaatcctcactaacagtgcccccaggaggaacactctcacct gtgcctcgaacactggcacagtcaccaatgtcagtatccttactggtttcagcagaaccc tagtcaagtccccagggcattgacttaggatacaagcaataaacacttctggatccctac caagctttcagtttccctccttggatgtaaaactcccctgaccttctctggttccctagc ctgaggccaaggctgattaccactggtgggtactcatagtggtgctgca

SEQ ID NO. 349 IGLV7-38-2 (P)

>IGLV7-38-2*01 I Canis lupus familiaris boxer | P | V-REGION | caggtcatggtgactcaggagccttcatggccatgtccccaggagggacagtcactctca cctatgcctccagcacaggacactatccatactggatccaagaaaatattggccaagtca gggccatttatttataataaaaacaacaaatactgatttctcatgctcccttcttgggag caaatctgacatgaccatctcctagtgcccagcctgaggacgaggatgagtacccatggg ggctacactatagtggtgctggg

SEQ ID NO. 350 IGLV7-43-1 (P)

>IGLV7-43-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgactcaggagccttcatggtcgtgtccccaggagggacagtcactctca ctatgcctccagcacagaacactatccatactggatccaggaaaatattggccaagtcta gagcatttatttataaaagaaacaataaatactgatttctaggctcccttcttgggaata aatctgacttgaccatctgctagtgcgcagcctgaggacgaggctgagtacccctagggg ttacac

SEQ ID NO. 351 IGLV7-44-1 (P)

>IGLV7-44-l*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgatgactcaggagtcctcactaacagtgtccccaggagggacattcactctc acctgtgcctccagccactggcatagtaacaatgctcagtatccttcctggttttaccag aagcctggccaagttcccagggcattgatttaggatacaagcaatgaaaattcctggacc cccaccaagtgctcaggttccctttgtggagcaatattctcctgaccctctacagtgcct tggtgagaacatagctgagtggcactggtggctgcttttattgtgatgctgggtgc

SEQ ID NO. 352 IGLV7-84-2 (P)

>IGLV7-84-2*01 I Canis lupus familiaris boxer | P | V-REGION | caggctgtgatgactcaagagtcctcactaacagtgtccccaggagggacattcactctc acctgcgcctccagctactggcatagtaacaatgctcagtatccttactggttttagcag aatcctggccaagtccccagggcattgatttaggatacaagcaatgaacacacctggacc cccaccatgtgctcaggttccctttgtggagcaatattctcctgaccctctacagtgcct tggtgagaacatagctgagtggcactggtggctgcttttattgtgatg

SEQ ID NO. 353 IGLV7-90-2 (P)

>IGLV7-90-2*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtggcataggagccttcatggccatatccccaggagggacagtcactctca cctatccctccagcacaggacactatctatactggatctagtagcatactggccaagtct aggtcatttatttataataaaaacaataaatactcatagacctccactcatttctcaggc tcccatcttgggggcaaatctgactggattgtcccctagtgcccagcctgaggatgaggc tgagtaccgctggggctacactatggtggtgtggg

SEQ ID NO. 354 IGLV7-120-1 (P)

>IGLV7-120-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtggcataggagccttcatggccatatccccaggagggacagtcactctca cctatccctccagcacaggacactatctatactggatctagtagcatactggccaagtct aggtcatttatttataataaaaacaataaatactcatagacctccactcatttctcaggc tcccatcttgggggcaaatctgactggattgtcccctagtgcccagcctgaggatgaggc tgagtaccgctggggctacactatggtggtgtggg

SEQ ID NO. 355 IGLV8-36 (F)

>IGLV8-36*01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtgacccaggagccatcactctcagtgtctctgggagggacagtcaccctc acatgtggcctcagctccgggtcagtctctacaagtaactaccccaactggtcccagcag accccagggcaggctcctcgcacgattatctacaacacaaacagccgcccctctggggtc cctaatcgcttcactggatccatctctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggacgaggctgactactactgtgctctgggattaagtagtagtagtagtta

SEQ ID NO. 356 IGLV8-39 (F)

>IGLV8-39*01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtctctacaagtaaccaccctagctggtaccagcag acccaagggaaggctcctcgcatgcttatctacaacacaaacaaccgcccctctgggatc cctaattgcttctctggatccatctctgggaacaaagcctccctcaccatcacaggagcc cagcctgaggacgagactgactattactgtttattgtatatgggtagtaacattta

SEQ ID NO. 357 IGLV8-40 (P)

>IGLV8-40*01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgacccaggagccatcactctaagtttctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtccctacaagtaactaccccagctggtttcagcag accccaggccgggctcctagaacagttatctacaacacaaacagctgcccctctggggtc cctaatcgcttcactggatccatctctggcaacaaagccgccctcaccatcacaagagcc cagcctgaggatgaggctgactcctgctgtgctgaatatcaaagcagtgggagcagctac acttacc

SEQ ID NO. 358 IGLV8-43 (P)

>IGLV8-43*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtaacccaggaacoatcactctcagtgtctcoatgagggacagtcacaetc acatgtggcctcagctctgggtcagtctctacaagtaactaccccaactggtaccagcag acccaaggccgggctcctcacagggttatctacaacacaaacaaccgcccctctggggtc cctgatcgcttctctggatccatctctgggaacaaagccgccctcaccatcacagctgcc cagcctgaggacgaggctgactattactgttcattgtatatgggtagtaacatttg

SEQ ID NO. 359 IGLV8-60 (P)

>IGLV8-60*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtgatcacccaagatacatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtctctacaagtaactaccccagctggtaccagcag acccaaggccgggatcctcgcatgcttatctacagcacaaacagccacccctctggggtc cctaattgcttcactagatccatctctgggaagaaagctgccctcaccatcacaggagcc cagcctgaggatgagactattattgttcactaaatatgggtagtacatgta

SEQ ID NO. 360 IGLV8-71 (P)

>IGLV8-71*01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgacccaggacccatcactgtcagtgtctagaggagggacagtcacactc acttgtggcctcagctctgggtcagtcactacaataaataccccagctggtcccagcaga ccccagggcaggctcctcgcatgattatctatgacacaaacagccgcccctctggggtcc ctgatcgcttctctggatccatctgtgggaacaaagctgccctcaccatcacaggagccc atcctgaggatgagactgactactactgtggtatacaacatggcagtgggagcagcctca cttacc SEQ ID NO. 361 IGLV8-74-1 (ORF)

>IGLV8-74-l*01 I Canis lupus familiaris boxer | ORF | V-REGION | cagattgtggtgacccaggagccatcactgtcagtgtctccaggaggaacagttacactc acatgtggcctaagctctgggtcagtcactataagtaactaccctgattggtaccagcag actccaggcaggtctcctcgcatgcttatctacaacacaaacaaccgcccctctggggtc cctaatcacttctctggatccatctctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggatgaggcttactactactgtgctgtgtatcaaggcagtgggagcagctac acttacc

SEQ ID NO. 362 IGLV8-76-1 (P)

>IGLV8-76-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcacccaggatcoatcactctcagtgtctccaggaggaacagtcacaetc acatgtggcctcagctctgggtcagtctctacaagtaactaccccggctggtaccagcag acccaagtgaaagctccttgcatgcttatctacagcacaaacagctacccctctggggtt cctaattgcttcactggatccatctctgggaagaaagctgccctcaccatcacaggagac cagcctgaggatgagactattattgttcactgcatatgggtagtacactta

SEQ ID NO. 363 IGLV8-88-4 (P)

>IGLV8-88-4*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtggctcaggagtcatcagtctcagtgtctccaggagggacagtcacactc acttgtggcctcagctctgggtcagtgactacaagtaactaccacagctggtaccagcgg acccaaggccggtctcctcacatgcttatctatgacacaagcagccgtccttctgaggtc ctgatcgcttccctggttccatctctgggaacaaagctgccctcactgtcagaggagccc agcctgaggacgaggctgactactactgtggcatgcatgatgtcagtgggaggaattaca attacc

SEQ ID NO. 364 IGLV8-89-3 (P)

>IGLV8-89-3*01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtggccaggaggcattgttgtcagtgtctccaggagggagagtcacactca cttgtggcctcagctctgggtcagtcactacaagtaactaccccaactggttccagcaga ccccagggcgggctcctggcacgattatctacagcacaaaagactgcccctctggggtcc ctgactgcttctctagatccatctctgggaacaaagccgccctcaccatcacaggagccc agtctgaggacgaggctattactgttttacacgacatggtagtgggagctgctacactta cc

SEQ ID NO. 365 IGLV8-90 (P)

>IGLV8-90*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acttgtggcctcagctctgggtcagtctctacaggtaacaaacctggctggtaccagcac accccaggccaggctcctcgcaggattatctatgacacaagcagccgcccttctggggtc cctgatcgcttctctggatccatctctgagaacaaaactgccctcaccatcacagaagcc caacctgaggatgaggctgactacatcatatatgagtggtggtgctta

SEQ ID NO. 366 IGLV8-90-1 (P)

>IGLV8-90-l*01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgacccaggaggcatcgttgttagtgtctcctggagggatagtcacactc acttgtggcctcagctctggatcaatcactacaagtaactaccccaactggctccagcag accccagggcgggctcctcgcagatgatctatggcacaaaaagccgcccctctggggtcc ctgatcgcttctgtagatccatctctgggaacaaagccgccctcaccatcacaggagccc agtctgaggatgaggctgactattactgttttacacgacatggcagtgggagcagctaca attac

SEQ ID NO. 367 IGLV8-90-3 (P)

>IGLV8-90-3*01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtgacccaggagtcatcagtctcagtgtctccaggaggaacagtcacactc ccttgtggcctcagctctgggtcactgactacaagtaacactacaccagctggtaccagc agacccaaggccagtctcctcgcatgcttgtctatgacacaagcagctgtccctctgagg ttcctgatcacttctctggatccatttctgggaacaaagccaccctcaccatcacaggag cccagcctgaggacgaggctgactactactgtggcatgcatgatgtcagtgggagcagct aaaattacc SEQ ID NO. 368 IGLV8-90-4 (P)

>IGLV8-90-4*01 I Canis lupus familiaris boxer | P | V-REGION | catattttggtgactcaggagccatcactgtcagtgtctccatgagggacagtcacactc acttgtggcctcagctctgggtcagtcactacaagtaactaccccaggtataccagcaga acccaggcaaggctcctagcacagttatctacaacaaaaacagctgcccctctggggtcc atggtcgattctctggatccatctctggaagcaaagccgccttcacaatcacaggagccc agcctgaggttgaggctgactactactgtgttacagaacatggctcctcacatgggaaca gcctcactcac

SEQ ID NO. 369 IGLV8-92-1 (P)

> IGLV8-92-1* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcacccaggatccgtcactctcagtgtctccaggagggacagtcacattc acatgtggcctcagctctgggtaagtctctacaagaaactaccccagctggtaccagcag acccaaggccaggctccttgcatgcttatctacagcacaagcagacacccttctggggtc cctgatcgcttctctggatccatctctgggaacaaagtcgccctcaccatcacaggagcc cagcctgaggataagactattattgttcactgcatatgggtagtacattta

SEQ ID NO. 370 IGLV8-93 (F)

>IGLV8-93*01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag acccaaggccgggctcctcgcacgattatctacaacacaagcagccgcccctctggggtc cctaatcgcttctctggatccatctctggaaacaaagccgccctcaccatcacaggagcc cagcccgaggatgaggctgactattactgttccttgtatacgggtagttacactga

SEQ ID NO. 371 IGLV8-99 (F)

>IGLV8-99*01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtcacccagaagccatcactctcagtgtctccaggagggacagtcacactc atatgtggcttcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag acccaaggccgggcttctcgcacaattatctacagcacaagcagccgcccctctggggtc cctaatcgcttccctggatccatctctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggacgaggctgactattactgttccttgtatatgggtagttacactga

SEQ ID NO. 372 IGLV8-102 (ORF)

>IGLV8-102* 01 I Canis lupus familiaris boxer | ORF | V-REGION | cagattgtagtgacccaggaaccatcactgtctccaggagggacagtcctactcacttgt ggcctcagctctgggtcagtcactacaagtaactactccagctggtaccagcagacccca gggcgggctcctcgcacgattatctacaacactaacagccacccctctggagtccctgat cgcttctctggatccatctctgggaacaaagcggcgctcaccatcacaggagcccagcct gaggacgaggctgactactactgtgttacagaacatggtagtgggagcagcttcacttac

SEQ ID NO. 373 IGLV8-108 (F)

>IGLV8-108* 01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtgactcaggagtcatcagtctcagtgtctccaggagggacagtcacactc acgtgtgacctcagctctgggtcagtgactacaagtaacaaccccagctggtaccagcag acccaaggccgatctcctcgcatgcttatctatgacacaagcagctgtccctcggaggtc cctgatcgcttctctggatccatttctgggaacacagctgccctcaccatcacaggagcc cagcctgaggacaaggctgactactactgtagtatgcatgatgtcagtgggagcagctac aattacc

SEQ ID NO. 374 IGLV8-113 (P)

>IGLV8-113* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcacccaggagccatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagttctgggtcagtcactataagtaactaccccagctggtcccagcag accccagggcaggctcctcacacaataatctacaggacaaacagctgaccctctggggtc cctgatcgcttctctggatccatctctgggaacaacgccgccctcagcatcacagtcgcc cagcctgaggacgaggctgactattactgttcattgtatatgggtagtaacattta

SEQ ID NO. 375 IGLV8-113-3 (P)

>IGLV8-113-3* 01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgacccaggagccatcactctcagtgtctagaggagggacagtcacactc acttgtggcctcagctctgagtcaatcactacaactaccccagctgatcccagcagaccc cagggcaggctcctcacacaattatctatgacaaaaacagccgcccctctggggtccctg atcacttctcaggatccatctgtgggaacaaagccaccctcaccatcacaggaacccagc ctgaggacaaggctgactactactgtggtatccaacatggcagtaggaggagcctcatta acc

SEQ ID NO. 376 IGLV8-117 (P)

>IGLV8-117* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgttgtgactcaggagtcatcagtctcagtgtctccaggagggacagtaacactc acgtgtagcctcagctctgggtcagtgactacaagtaagtactccagctggaccagtaga cccaaggccgatctcctcgcatgcttatctatgacacaagcagccgtccctctgaggtcc ctgatcgcttctctggatccatctccgggaacaaagctgccctcaccatcacaggagccc agcctgaggacgaggctgactactactgtggtatgcatgatgtcagtgggaggagttaca attacc

SEQ ID NO. 377 IGLV8-118-3 (P)

>IGLV8-118-3* 01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtggccaggaggcattgttgtcagtgtcctctggagggagagtcacactca cttgtggcctcagctctgggtcagtcactacaagtaactaccccaactggttccagcaga ccccagggcgggctcctggcacgattatgtacagcacaaaagactgcccctctggggtcc ctgattgcttctctagatccatctctgggaacaaagccgccctcaccatcacaggagccc agtctgaggacgaggttattactgttttacacgacatggtagtgggagctgctacactta cc

SEQ ID NO. 378 IGLV8-119 (P)

>IGLV8-119* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acttgtggcctcagctctgggtcagtctctacaggtaacaaacctggctggtaccagcac accccaggccaggctcctcgcaggattatctatgacacaagcagccgcccttctggggtc cctgatcgcttctctggatccatctctgagaacaaagctgccctcaccatcacagaagcc cagcctgaggatgaggctgcctaccactgttcgctgtatatgagtggtggtgctta

SEQ ID NO. 379 IGLV8-120 (P)

>IGLV8-120* 01 I Canis lupus familiaris boxer | P | V-REGION | cagattgtggtgacccaggaggcatcgttgtcagtgtctcctggagggatagtcacactc acttgtggcctcagctctggatcaatcactacaagtaactaccccaactggttccagcag accccagggcgggctcctcgcagatgatctatggcacaaaaagccgcccctctggggtcc ctgatcgcttctgtagatccatctctgggaacaaagccgccctcaccatcacaggagccc agtctgaggatgaggctgactattactgttttacacgacatggcagtgggagcagctaca attacc

SEQ ID NO. 380 IGLV8-121 (P)

>IGLV8-121* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtgacccaggagtcatcagtctcagtgtctccagtcggaacagtcacactc acttgtggcctcagctctgggtcactgactacaagtaactacaccagctggtaccagcag acccaaggccagtctcctcgcatgcttgtctatgacacaagcagctgtccctctgaagtt cctgatcacttctctggatccatttctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggacgaggctgactactactgtggtatgcatgatgtcagtgggagcagctaa aattacc

SEQ ID NO. 381 IGLV8-121-1 (P)

>IGLV8-121-1* 01 I Canis lupus familiaris boxer | P | V-REGION | catattttggtgactcaggagccatcactgtcagtgtctccatgagggacagtcacactc acttgtggcctcagctctgggtcagtcactacaagtaactaccccaggtataccagcaga acccaggcaaggctcctagcacagttatctacaacaaaaacagctgcccctctggggtcc atggtcgattctctggatccatctctggaagcaaagccgccttcacaatcacaggagccc agcctgaggttgaggctgactactactgtgttacagaacatggctcct

SEQ ID NO. 382 IGLV8-124 (P)

>IGLV8-124* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcaaccaggatccgtcactctcagtgtctccaggagggacagtcacattc acatgtggcctcagctctgggtaagtctctgcaagaaactaccccagctggtaccagcag acccaaggccaggctccttgcatgcttatctacagcacaagcagccgcccttctggggtc cctgatcgcttctctggatccatctctgggaacaaagtcgccctcaccatcacaggagcc cagcctgaggatgagactattattgttcactgcatatgggtagtacattta

SEQ ID NO. 383 IGLV8-128 (F)

>IGLV8-128* 01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag accctaggccgggctcctcgcacgattatctacagaacaagcagccgcccctctggggtc cctaatcgcttctctggatccatctctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggacgaggctgactattactgttccttgtatatgggtagttacactga

SEQ ID NO. 384 IGLV8-137 (P)

>IGLV8-137* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcaccaaggatccatcactctcagtgtctccaggagggacagtcacattc acatgtggcctcagctctgggtcagtctttacaagtaactaccccagctggtaccagcag acccatggccgggctcctcgcatgcttatctacagcacaaggagctgcccccccggggtc cctgatcgcttctctggatccatctctgggaacaaagttgccctcaccatcacaggagcc cagcctgaggatgagactattattgttcactgtgtatgggtagtacattta

SEQ ID NO. 385 IGLV8-142 (F)

>IGLV8-142* 01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtcacccagaagccatcactctcagtgtctccaggagggacagtcacactc atatgtggcctcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag acccaaggccgggcttctcgcacaattatctacagcacaagcagccgcccctctggggtc cctaatcgcttcactggatccatctctgggaacaaagccgccctcaccatcacaggagcc cagcctgaggacgaggctgactattactgttccttgtatatgggtagttacactga

SEQ ID NO. 386 IGLV8-150-1 (ORF)

>IGLV8-150-1* 01 I Canis lupus familiaris boxer | ORF | V-REGION | cagattgtggtgacccaggaacoatcactgtcagtgtctccaggagggacactcacaetc acttgtggcctcagctctgggtcagtcactacaagtaactaccccagctggtaccagcag accccaggccaggctcctagcacagttatctacaacacaaacagccgcccctctggtgtc cctgatcacttctctggatccgtctctgggaacaaagccgccctcatcatcacaggagcc cagcctgaggacgaggctgatgactactctgttgcagaacatgtcagtgggagcagcttc acttacc

SEQ ID NO. 387 IGLV8-153 (F)

>IGLV8-153* 01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtaacccaggagccatcactctcagtgtctccaggagggacagtcacactc acatgtggcctcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag acccaaggccgggctcctcgcacgattatctacaacacaagcagccgcccctctggggtc cctaatcgcttctctggatccatctctggaaacaaagccgccctcaccatcacaggagcc cagcccgaggatgaggctgactattactgttccttgtatacgggtagttacactga

SEQ ID NO. 388 IGLV8-156 (P)

>IGLV8-156* 01 I Canis lupus familiaris boxer | P | V-REGION | cagactgtggtcaccaaggatccatcactctcagtgtttccaggagggacagtcacattc acatgtggcctcagctctgggtcagtctttacaagtaactaccccagctggtaccagcag acccatggccgggctcctcgcatgcttatctacagcacaagcagctgcccccccggggtc cctgatcgcttctctggatccatctctgggaacaaagttgccctcaccatcacaggagcc cagcctgaggatgagactattattgttcactgtgtatgggtagtacattta

SEQ ID NO. 389 IGLV8-161 (F)

>IGLV8-161* 01 I Canis lupus familiaris boxer | F | V-REGION | cagactgtggtcacccagaagccatcactctcagtgtctccaggagggacagtcacactc atatgtggcctcagctctgggtcagtctctacaagtaattaccctggctggtaccagcag acccaaggccgggcttctcgcacaattatctacagcacaagcagccgcccctctggggtc cctaatcgcttccctggatccatctctgggaacaaagccgccctcatcatcacaggagcc cagcctgaggacgaggctgactattactgttccttgtatatgggtagttacactga Germline Gl sequences

SEQ ID NO. 390 IGLJ1 (F)

>IGLJl* 01 I Canis lupus familiaris boxer | F | J-REGION |

ttgggtattcggtgaagggacccagctgaccgtcctcg

SEQ ID NO. 391 IGLJ2 (F)

>IGLJ2* 01 I Canis lupus familiaris boxer | F | J-REGION |

tatggtattcggcagagggacccagctgaccatcctcg

SEQ ID NO. 392 IGLJ3 (F)

>IGLJ3* 01 I Canis lupus familiaris boxer | F | J-REGION |

tagtgtgttcggcggaggcacccatctgaccgtcctcg

SEQ ID NO. 393 IGLJ4 (F)

>IGLJ4* 01 I Canis lupus familiaris boxer | F | J-REGION | |

ttacgtgttcggctcaggaacccaactgaccgtccttg

SEQ ID NO. 394 IGLJ5 (F)

>IGLJ5* 01 I Canis lupus familiaris boxer | F | J-REGION |

tattgtgttcggcggaggcacccatctgaccgtcctcg

SEQ ID NO. 395 IGLJ6 (F)

>IGLJ6* 01 I Canis lupus familiaris boxer | F | J-REGION |

tggtgtgttcggcggaggcacccacctgaccgtcctcg

SEQ ID NO. 396 IGLJ7 (F)

>IGLJ7* 01 I Canis lupus familiaris boxer | F | J-REGION |

tgctgtgttcggcggaggcacccacctgaccgtcctcg

SEQ ID NO. 397 IGLJ8 (F)

>IGLJ8* 01 I Canis lupus familiaris boxer | F | J-REGION |

tgctgtgttcggcggaggcacccacctgaccgtcctcg

SEQ ID NO. 398 IGLJ9 (F)

>IGLJ9* 01 I Canis lupus familiaris boxer | F | J-REGION |

ttacgtgttcggctcaggaacccaactgaccgtccttg

Table 4. Canine constant region genes

IGHC sequences

Functionality is shown between brackets, [F] and [P], when the accession number (underlined) refers to rearranged genomic DNA or cDNA and the corresponding germline gene has not yet been isolated.

SEQ ID NO. 399 IGHA (F)

>IGHA*01 I Canis lupus familiaris boxer |F| CHI |

nagtccaaaaccagccccagtgtgttcccgctgagcctctgccaccaggagtcagaa ggg

tacgtggtcatcggctgcctggtgcagggattcttcccaccggagcctgtgaacgtg acc

tggaatgccggcaaggacagcacatctgtcaagaacttcccccccatgaaggctgct acc

ggaagcctatacacoatgagcagccagttgaccctgccageegcccagtgccctgat gac

tcgtctgtgaaatgccaagtgcagcatgcttccagccccagcaaggcagtgtctgtg ccc

tgcaaa

>IGHA*01 I Canis lupus familiaris boxer | F | H-CH2 |

gataactgtcatccgtgtcctcatccaagtccctcgtgcaatgagccccgcctgtca cta cagaagccagccctcgaggatctgcttttaggctccaatgccagcctcacatgcacactg agtggcctgaaagaccccaagggtgccaccttcacctggaacccctccaaagggaaggaa cccatccagaagaatcctgagcgtgactcctgtggctgctacagtgtgtccagtgtccta ccaggctgtgctgatccatggaaccatggggacaccttctcctgcacagccacccaccct gaatccaagagcccgatcactgtcagcatcaccaaaaccaca

>IGHA*01 I Canis lupus familiaris boxer | F | CH3-CHS |

gagcacatcccgccccaggtccacctgctgccgccgccgtcggaagagctggccctc aat gagctggtgacactgacgtgcttggtgaggggcttcaaaccaaaagatgtgctcgtacga tggctgcaagggacccaggagctaccccaagagaagtacttgacctgggagcccctgaag gagcctgaccagaccaacatgtttgccgtgaccagcatgctgagggtgacagccgaagac tggaagcagggggagaagttctcctgcatggtgggccacgaggctctgcccatgtccttc acccagaagaccatcgaccgcctggcgggtaaacccacccacgtcaacgtgtctgtggtc atggcagaggtggacggcatctgctac

>IGHA*01 I Canis lupus familiaris boxer|F|M|

gactcacagtgtcttgcaggttaccgggagccacttccctggctggtgctggacctg tcg caggaggacctggaggaggatgccccaggagccagcctgtggcccactaccgtcaccctt ctcaccctcttcctactgagtctcttctacagcacagcactgactgtgacaagcgtgcgg ggcccaactgacagcagagagggcccccagtac

SEQ ID NO. 400 IGHD (ORF)

> I IGHD* 01 I Canis lupus familiaris boxer | ORF | CHI |

gaatcgtcacttctgctccccttggtctcaggatgtaaggtcccaaaaaatggtgag gac ataaccctggcctgcttggcaaaaggacccttcctagattctgtgcgggtcacgacaggc ccagagtcacaggcccagatggaaaagaccacactgaagatgctaaagataccggaccac actcaggtgtctctcctgtccaccccctggaaaccaggcctgcactactgcgaagccatc aggaaagataacaaagagaagctgaagaaagccatccactggcca

>IGHD*01 I Canis lupus familiaris boxer | ORF | HI |

gcatcctgggaaactgctatctccctgttgactcatgcgccatcccgaccccaggac cac acccaagcccccagcatggccagggtctca

>IGHD*01 I Canis lupus familiaris boxer | ORF | H2 |

gtgcctcccaccagccacacccagacgcaagcccaggagccaggatgcccagtggac acc atcctcaga

>IGHD*01 I Canis lupus familiaris boxer | ORF | CH2 |

gagtgttggaaccacacccaccctcccagcctctacatgctgcgccctcccctgcgg gga ccatggctccagggagaagctgctttcacctgcctggtggtgggagatgaccttcagaag gcccacctgtcctgggaggtagccggggcgccccccagcgaggctgtggaggagaggcca ctgcaggagcatgagaatggctcccagagctggagcagccgcctggtcttgcccatatcc ctgtgggcctcaggagccaacatcacctgcacgctgagcctccccagcatgccttcccag gtggtgtccgcagcagccagagagoat

>IGHD*01 I Canis lupus familiaris boxer | ORF | CH3 |

gctgccagagcacccagcagcctcaatgtccatgccctgaccatgcccagagcagcc tcc tggttcctgtgcgaggtgtccggcttctcaccccctgacatcctcctcacctggatcaag gaccagattgaggtggacccttcttggttcgccactgcaccccccatggcccagccgggc agtggcacgttccagacctggagtctcctgcgtgtcctcgctccccagggccctcacccg cccacctacacgtgtgtagtcaggcacgaggcctcccggaagctgctcaacaccagctgg agcctggacagt

>IGHD*01 I Canis lupus familiaris boxer | ORF | Ml |

ggtctgaccatgacccccccagcccctcagagccacgacgagagcagcggggactcc atg gatctggaagatgccagcggactgtggcccacgttcgctgccctcttcgtcctcactctg ctctacagcggcttcgtcaccttcctcaaa

>IGHD*01 I Canis lupus familiaris boxer | ORF | M2 |

gtgaag

SEQ ID NO. 401 IGHE (F)

>IGHE*01 I Canis lupus familiaris boxer |F| CHI |

nccaccagccaggacctgtctgtgttccccttggcctcctgctgtaaagacaacatc gcc agtacctctgttacactgggctgtctggtcaccggctatctccccatgtcgacaactgtg acctgggacacggggtctctaaataagaatgtcacgaccttccccaccaccttccacgag acctacggcctccacagcatcgtcagccaggtgaccgcctcgggcgagtgggccaaacag aggttcacctgcagcgtggctcacgctgagtccaccgccatcaacaagaccttcagt

>IGHE*01 I Canis lupus familiaris boxer|F|CH2|

gcatgtgccttaaacttcattccgcctaccgtgaagctcttccactcctcctgcaac ccc gtcggtgatacccacaccaccatccagctcctgtgcctcatctctggctacgtcccaggt gacatggaggtcatctggctggtggatgggcaaaaggctacaaacatattcccatacact gcacccggcacaaaggagggcaacgtgacctctacccacagcgagctcaacatcacccag ggcgagtgggtatcccaaaaaacctacacctgccaggtcacctatcaaggctttaccttt aaagatgaggctcgcaagtgctca

>IGHE*01 I Canis lupus familiaris boxer |F|CH3|

gagtccgacccccgaggcgtgagcagctacctgagcccacccagcccccttgacctg tat gtccacaaggcgcccaagatcacctgcctggtagtggacctggccaccatggaaggcatg aacctgacctggtaccgggagagcaaagaacccgtgaacccgggccctttgaacaagaag gatcacttcaatgggacgatcacagtcacgtctaccctgccagtgaacaccaatgactgg atcgagggcgagacctactattgcagggtgacccacccgcacctgcccaaggacatcgtg cgctccattgccaaggcccct

>IGHE*01 I Canis lupus familiaris boxer | F | CH4-CHS |

ggcaagcgtgcccccccggatgtgtacttgttcctgccaccggaggaggagcagggg acc aaggacagagtcaccctcacgtgcctgatccagaacttcttccccgcggacatttcagtg caatggctgcgaaacgacagccccatccagacagaccagtacaccaccacggggccccac aaggtctcgggctccaggcctgccttcttcatcttcagccgcctggaggttagccgggtg gactgggagcagaaaaacaaattcacctgccaagtggtgcatgaggcgctgtccggctct aggatcctccagaaatgggtgtccaaaacccccggtaaa

>IGHE*01 I Canis lupus familiaris boxer |F|M1|

gagctccaggagctgtgcgcggatgccactgagagtgaggagctggacgagctgtgg gcc agcctgctcatcttcatcaccctcttcctgctcagcgtgagctacggcgccaccagcacc ctcttcaag

>IGHE*01 I Canis lupus familiaris boxer |F|M2|

gtgaagtgggtactcgccaccgtcctgcaggagaagccacaggccgcccaagactac gcc aacatcgtgcggccggcacag

SEQ ID NO. 402 IGHG1 [F]

>AF354264 I IGHG1*01 I Canis lupus familiaris | (F) I CHI | |

gcctccaccacggccccctcggttttcccactggcccccagctgcgggtccacttcc ggc tccacggtggccctggcctgcctggtgtcaggctacttccccgagcctgtaactgtgtcc tggaattccggctccttgaccagcggtgtgcacaccttcccgtccgtcctgcagtcctca gggcttcactccctcagcagcatggtgacagtgccctccagcaggtggcccagcgagacc ttcacctgcaacgtggtccacccagccagcaacactaaagtagacaagcca

>IGHG1* 01 I Canis lupus familiaris | (F) |H|

gtgttcaatgaatgcagatgcactgatacacccccatgccca

>IGHG1* 01 I Canis lupus familiaris | (F) | CH2 |

gtccctgaacctctgggagggccttcggtcctcatctttcccccgaaacccaaggac atc ctcaggattacccgaacacccgaggtcacctgtgtggtgttagatctgggccgtgaggac cctgaggtgcagatcagctggttcgtggatggtaaggaggtgcacacagccaagacccag tctcgtgagcagcagttcaacggcacctaccgtgtggtcagcgtcctccccattgagcac caggactggctcacagggaaggagttcaagtgcagagtcaaccacatagacctcccgtct cccatcgagaggaccatctctaaggccaga

>IGHG1* 01 I Canis lupus familiaris | (F) | CH3-CHS |

gggagggcccataagcccagtgtgtatgtcctgccgccatccccaaaggagttgtca tcc agtgacacagtcagcatcacctgcctgataaaagacttctacccacctgacattgatgtg gagtggcagagcaatggacagcaggagcccgagaggaagcaccgcatgaccccgccccag ctggacgaggacgggtcctacttcctgtacagcaagctctctgtggacaagagccgctgg cagcagggagaccccttcacatgtgcggtgatgcatgaaactctacagaaccactacaca gatctatccctctcccattctccgggtaaa

SEQ ID NO. 403 IGHG2 (F)

>IGHG2* 01 I Canis lupus familiaris boxer|F|CHl|

ncctccaccacggccccctcggttttcccactggcccccagctgcgggtccacttcc ggc tccacggtggccctggcctgcctggtgtcaggctacttccccgagcctgtaactgtgtcc tggaattccggctccttgaccagcggtgtgcacaccttcccgtccgtcctgcagtcctca gggctctactccctcagcagcatggtgacagtgccctccagcaggtggcccagcgagacc ttcacctgcaacgtggcccacccggccagcaaaactaaagtagacaagcca

> I IGHG2*01 I Canis lupus familiaris boxer |F|H|

gtgcccaaaagagaaaatggaagagttcctcgcccacctgattgtcccaaatgccca >IGHG2* 01 I Canis lupus familiaris boxer |F|CH2|

gcccctgaaatgctgggagggccttcggtcttcatctttcccccgaaacccaaggac acc ctcttgattgcccgaacacctgaggtcacatgtgtggtggtggatctggacccagaagac cctgaggtgcagatcagctggttcgtggacggtaagcagatgcaaacagccaagactcag cctcgtgaggagcagttcaatggcacctaccgtgtggtcagtgtcctccccattgggcac caggactggctcaaggggaagcagttcacgtgcaaagtcaacaacaaagccctcccatcc ccgatcgagaggaccatctccaaggccaga

>IGHG2* 01 I Canis lupus familiaris boxer | F | CH3-CHS |

gggcaggcccatcaacccagtgtgtatgtcctgccgccatcccgggaggagttgagc aag aacacagtcagcttgacatgcctgatcaaagacttcttcccacctgacattgatgtggag tggcagagcaatggacagcaggagcctgagagcaagtaccgcacgaccccgccccagctg gacgaggacgggtcctacttcctgtacagcaagctctctgtggacaagagccgctggcag cggggagacaccttcatatgtgcggtgatgcatgaagctctacacaaccactacacacag aaatccctctcccattctccgggtaaa

>IGHG2* 01 I Canis lupus familiaris boxer | F | Ml |

gagctgatcctggatgacagctgtgctgaggaccaggacggggagctggacgggctg tgg accaccatctccatcttcatcaccctcttcctgctcagcgtgtgctacagcgccactgtc accctcttcaag

> I IGHG2*01 I Canis lupus familiaris boxer |F|M2|

gtgaagtggatcttctcatcagtggtggagctgaagcgcacgattgtccccgactac agg aatatgatcgggcagggggcc

SEQ ID NO. 404 IGHG3 [F]

>AF35_42_66 I IGHG3* 01 I Canis lupus familiaris | (F) I CHI | gcctccaccacggccccctcggttttcccactggcccccagctgtgggtcccaatccggc tccacggtggccctggcctgcctggtgtcaggctacatccccgagcctgtaactgtgtcc tggaattccgtctccttgaccagcggtgtgcacaccttcccgtccgtcctgcagtcctca gggctctactccctcagcagcatggtgacagtgccctccagcaggtggcccagcgagacc ttcacctgcaatgtggcccacccggccaccaacactaaagtagacaagcca

>IGHG3* 01 I Canis lupus familiaris | (F) |H|

gtggccaaagaatgcgagtgcaagtgtaactgtaacaactgcccatgccca

>IGHG3* 01 I Canis lupus familiaris | (F) | CH2 |

ggttgtggcctgctgggagggccttcggtcttcatctttcccccaaaacccaaggac atc ctcgtgactgcccggacacccacagtcacttgtgtggtggtggatctggacccagaaaac cctgaggtgcagatcagctggttcgtggatagtaagcaggtgcaaacagccaacacgcag cctcgtgaggagcagtccaatggcacctaccgtgtggtcagtgtcctccccattgggcac caggactggctttcagggaagcagttcaagtgcaaagtcaacaacaaagccctcccatcc cccattgaggagatcatctccaagacccca

>IGHG3* 01 I Canis lupus familiaris | (F) | CH3-CHS |

gggcaggcccatcagcctaatgtgtatgtcctgccgccatcgcgggatgagatgagc aag aatacggtcaccctgacctgtctggtcaaagacttcttcccacctgagattgatgtggag tggcagagcaatggacagcaggagcctgagagcaagtaccgcatgaccccgccccagctg gatgaagatgggtcctacttcctatacagcaagctctccgtggacaagagccgctggcag cggggagacaccttcatatgtgcggtgatgcatgaagctctacacaaccactacacacag atatccctctcccattctccgggtaaa

SEQ ID NO. 405 IGHG4 [F]

>AF354267 I IGHG4*01 I Canis lupus familiaris | (F) I CHI | gcctccaccacggccccctcggttttcccactggcccccagctgcgggtccacttccggc tccacggtggccctggcctgcctggtgtcaggctacttccccgagcctgtaactgtgtcc tggaattccggctccttgaccagcggtgtgcacaccttcccgtccgtcctgcagtcctca gggctctactccctcagcagcacggtgacagtgccctccagcaggtggcccagcgagacc ttcacctgcaacgtggtccacccggccagcaacactaaagtagacaagcca

>IGHG4* 01 I Canis lupus familiaris | (F) |H|

gtgcccaaagagtccacctgcaagtgtatatccccatgccca

>IGHG4* 01 I Canis lupus familiaris | (F) | CH2 |

gtccctgaatcactgggagggccttcggtcttcatctttcccccgaaacccaaggac atc ctcaggattacccgaacacccgagatcacctgtgtggtgttagatctgggccgtgaggac cctgaggtgcagatcagctggttcgtggatggtaaggaggtgcacacagccaagacgcag cctcgtgagcagcagttcaacagcacctaccgtgtggtcagcgtcctccccattgagcac caggactggctcaccggaaaggagttcaagtgcagagtcaaccacataggcctcccgtcc cccatcgagaggactatctccaaagccaga

>IGHG4* 01 I Canis lupus familiaris | (F) | CH3-CHS |

gggcaagcccatcagcccagtgtgtatgtcctgccaccatccccaaaggagttgtca tcc agtgacacggtcaccctgacctgcctgatcaaagacttcttcccacctgagattgatgtg gagtggcagagcaatggacagccggagcccgagagcaagtaccacacgactgcgccccag

ctggacgaggacgggtcctacttcctgtacagcaagctctctgtggacaagagccgc tgg

cagcagggagacaccttcacatgtgcggtgatgcatgaagctctacagaaccactac aca

gatctatccctctcccattctccgggtaaa

SEQ ID NO. 406 IGHM (F)

>IGHM*01 I Canis lupus familiaris boxer |F| CHI |

nagagtccatcccctccaaacctcttccccctcatcacctgtgagaactccctgtcc gat

gagaccctcgtggcoatgggctgcctggcccgggacttcctgcctggctcoatcacc ttc

tcctggaagtacgagaacctcagtgcaatcaacaaccaggacattaagaccttccct tca

gttctgagagagggcaagtatgtggcgacctctcaggtgttcctgccctccgtggac atc

atccagggttcagacgagtacatcacatgcaacgtcaagcactccaatggtgacaaa tct

gtgaacgtgcccatcaca

>IGHM*01 I Canis lupus familiaris boxer|F|CH2|

gggcctgtaccaacgtctcccaacgtgactgtcttcatcccaccccgcgacgccttc tct

ggcaatggccagcgcaagtcccagctcatctgccaggctgcaggtttcagccccaag cag

atttccgtgtcttggttccgtgatggaaagcagattgagtctggcttcaacacaggg aag

gcagaggccgaggagaaagagcatgggcctgtgacctacagcatcctcagcatgctg acc

atcaccgagagtgcctggctcagccagagcgtgttcacctgccacgtggagcacaat ggg

atcatcttccagaagaacgtgtcctccatgtgcacctcc

>IGHM*01 I Canis lupus familiaris boxer |F|CH3|

aatacacccgttggcatcagoatcttcaccatccccccctcctttgccagoatcttc aac

accaagtcagccaagctgtcctgcctggtcactgacctggccacttatgacagcctg acc

atctcctggacccgtcagaatggcgaggctctgaaaacccacaccaacatctctgag agc

catcccaacaacaccttcagtgccatgggggaagccactgtctgcgtggaggaatgg gag

tcaggcgagcagttcacctgcacagtgacccacacagatctgccctcaccgctgaag aag

accatctccaggcccaag

>IGHM*01 I Canis lupus familiaris boxer | F | CH4-CHS |

gatgtcaacaagcacatgccttctgtctacgtcctgcccccgagccgggagcagctg agc

ctgcgggaatcggcctcactcacctgcctggtgaaaggcttctcacccccagatgtg ttc

gtgcagtggctgcagaagggccagcccgtgccccctgacagctacgtgaccagcgcc ccg

atgcccgagccccaagcccccggcctctactttgtccacagcatcctgaccgtgagt gag

gaggactggaatgccggggagacctacacctgtgttgtaggccatgaggccctgccc cat

gtggtgaccgagaggagcgtggacaagtccaccggtaaacccaccttgtacaacgtg tcc

ctggtcttatctgacacagccagcacctgctac

>IGHM*01 I Canis lupus familiaris boxer |F|M1|

gggggggaggtgagtgccgaggaggaaggcttcgagaacctgaataccatggcatcc acc

ttcatcgtcctcttcctcctcagtgtcttctacagcaccacagtcactctgttcaag

>IGHM*01 I Canis lupus familiaris boxer |F|M2|

gtgaaa

IGKC sequences

SEQ ID NO. 407 IGKC (F)

>IGKC*01 I Canis lupus familiaris boxer | F | C-REGION |

cggaatgatgcccagccagccgtctatttgttccaaccatctccagaccagttacac aca

ggaagtgcctctgttgtgtgcttgctgaatagcttctaccccaaagacatcaatgtc aag

tggaaagtggatggtgtcatccaagacacaggcatccaggaaagtgtcacagagcag gac

aaggacagtacctacagcctcagcagcaccctgacgatgtccagtactgagtaccta agt

catgagttgtactcctgtgagatcactcacaagagcctgccctccaccctcatcaag agc

ttccaaaggagcgagtgtcagagagtggac

IGLC sequences

[F], Functionality defined for the available sequence of the gene (partial gene in 3' because of gaps in the sequence).

SEQ ID NO. 408 IGLC1 (F) >IGLC1* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaagtcctcccccttggtcacactcttcccgccctcctctgaggagctcggc gccaacaaggctaccctggtgtgcctcatcagcgacttctaccccagtggcctgaaagtg gcttggaaggcagatggcagcaccatcatccagggcgtggaaaccaccaagccctccaag cagagcaacaacaagtacacggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacgcaccaggggagcaccgtggagaagaaggtg gcccctgcagagtgctct

SEQ ID NO. 409 IGLC2 (F)

>IGLC2* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccctcagtcacactcttcccaccctcctctgaggagctcggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggcgtgacggtg gcctggaaggcagacggcagccccggcatccagggcgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacgcatgaggggagcaccgtggagaagaaggtg gcccccgcagagtgctct

SEQ ID NO. 410 IGLC3 (F)

>IGLC3* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctcggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagtggcgtgacggtg gcctggaaggcagacggcagccccgtcacccagggcgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacacacgaggggagcaccgtggagaagaaggtg gcccccgcagagtgctct

SEQ ID NO. 411 IGLC4 [F]

>IGLC4* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctcggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggtgtgacggtg gcctggaaggcagacggcagccccgtcacccagggcgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacacacgaggggagcactgtgg

SEQ ID NO. 412 IGLC5 (F)

>IGLC5* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccttcggtcacactcttcccgccctcctctgaggagcttggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggcgtgacagtg gcctggaaggcagacggcagccccatcacccagggtgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacgcacgaggggagcaccgtggagaagaaggtg gcccccgcagagtgctct

SEQ ID NO. 413 IGLC6 (F)

>IGLC6* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctcggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggtgtgacggtg gcctggaaggcagacggcagccccgtcacccagggcgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacgcacgaggggagcaccgtggagaagaaggtg gcccccgcagagtgctct

SEQ ID NO. 414 IGLC7 (F)

>IGLC7* 01 I Canis lupus familiaris boxer | F | C-REGION | ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctcggc gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggcgtgacggtg gcctggaaggcagacggcagccccgtcacccagggcgtggagaccaccaagccctccaag cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtggaaa tctcacagcagcttcagctgcctggtcacgcacgaggggagcaccgtggagaagaaggtg gcccccgcagagtgctct SEQ ID NO. 415 IGLC8 (F)

>IGLC8* 01 I Canis lupus familiaris boxer | F | C-REGION |

ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctc ggc

gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggcgtgacg gtg

gcctggaaggcagacggcagccccgtcacccagggcgtggagaccaccaagccctcc aag

cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtgg aaa

tctcacagcagcttcagctgcctggtcacgcacgaggggagcaccgtggagaagaag gtg

gcccccgcagagtgctct

SEQ ID NO. 416 IGLC9 (F)

>IGLC9* 01 I Canis lupus familiaris boxer | F | C-REGION |

ggtcagcccaaggcctccccctcggtcacactcttcccgccctcctctgaggagctc ggc

gccaacaaggccaccctggtgtgcctcatcagcgacttctaccccagcggcgtgacg gtg

gcctggaaggcagacggcagccccatcacccagggcgtggagaccaccaagccctcc aag

cagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgacaagtgg aaa

tctcacagcagcttcagctgcctggtcacgcacgaggggagcactgtggagaagaag gtg

gcccccgcagagtgctct

// End of canine Ig sequences

Table 5. PCR Primers

SEQ ID NO. 417 IF: ACATAATACACTGAAATGGAGCCC

SEQ ID NO. 418 1R : GTCCTTGGTCAACGTGAGGG

SEQ ID NO. 419 2F: CATAATACACTGAAATGGAGCCCT

SEQ ID NO. 420 2R : GCAACAGTGGTAGGTCGCTT

Table 6. Miscellaneous sequence data

Pre-DJ

This is a 21609 bp fragment upstream of the Ighd-5 DH gene. The pre-DJ sequence can be found in Mus musculus strain C57BL/6J chromosome 12, Assembly: GRCm38.p4, Annotation release 106, Sequence ID: NC_000078.6

The entire sequence lies between the two 100 bp sequences shown below:

Upstream of the Ighd-5 DH gene segment, corresponding to positions 113526905- 113527004 in NC 000078.6:

ATTTCTGTACCTGATCTATGTCAATATCTGTACCATGGCTCTAGCAGAGATGAAATA TG AGACAGTCTGATGTCATGTGGCCATGCCTGGTCCAGACTTG (SEQ ID NO. 421)

2 kb upstream of the ADAM6A gene corresponding to positions 113548415 - 113548514 in NC_000078.6:

GTCAATCAGCAGAAATCCATCATACATGAGACAAAGTTATAATCAAGAAATGTTGCCCA TAGGAAACAGAGGATATCTCTAGCACTCAGAGACTGAGCAC (SEQ ID NO. 422) ADAM6A

ADAM6A (a disintegrin and metallopeptidase domain 6A) is a gene involved in male fertility. The ADAM6A sequence can be found in Mus musculus strain C57BL/6J chromosome 12, Assembly: GRCm38.p4, Annotation release 106, Sequence ID:

NC_000078.6 at position 113543908-113546414.

ADAM6A sequence ID: OTTMUSG00000051592 (VEGA)

Table 7. Chimeric canine/mouse Ig gene sequences

Igk Version A

Sequence upstream of mouse Igkv 1-133 (SEQ ID NO: 423)

GCATTGAATAAACCAGTATAAACAAGCAAGCAAAGATAGATAGATAGATAGATAGATAGA TAGATAGATAC ATAGATAGATAGATAGATAGATAGATGATAGATAGATAGATAGATAGATAGATTTTTACG TATAATACAAT AAAAACATTCATTGTCCCTCTATTGGTGACTACTCAAGGAAAAAAATGTTCATATGCAAG AAAAAATGTTA TCATTACCAGATGATCCAGCAATCTAGCAATATATATATTGTTTATTCACAAAACATGAA TGAACCTTTTA AGAAGCTGTTACAGTGTAAAAATTAAGTTAAATCACTGAAGAACATATACTGTGTGATTT CATTCAAATGA AATTTGAGAAGTAAATATATATGTATATATATATATATGTAAAAAATATAAGTCTGAACT ACAAAAATTCA ATTTGTTTGATATGTAAGAATAAGAAAAATTGACCCCCAAAATTTGTTAATAATTAGGTA TGTGTATTTTT ATGAATATATAAGTATAATAATGCTTATAGTATACACTATTCTGAATCACATTTATTCCC TAAGTGTGTTC CCTTGATTATAATTAAAAGTATATTTTTTAAATACAGAGTCAGAGTACAGTCAATAAGGC GAAAATATAGT TGAATGATTTGCTTCAGCTTTTGTAATGTACTAGAGATTGTGAGTACAAAGTCTCAGAGC TCATTTTATCC CTGACAATAACCAGCTCTGTGCTTCAAGTACATTTCCATCTTTCTCTGAAATTTAGTCTT ATATAGATAGA CAAAATTTAAGTAAATTTCAAACTACACAGAACAACTAAGTTGTTGTTTCATATTGATAA TGGATTTGAAC TGCATTAACAGAACTTTAACATCCTGCTTATTCTCCCTTCAGCCATCATATTTTGCTTTA TTATTTTCACT TTTTGAGTTATTTTTCACATTCAGAAAGCTCACATAATTGTCACTTCTTTGTATACTGGT ATACAGACCAG AACATTTGCATATTGTTCCCTGGGGAGGTCTTTGCCCTGTTGGCCTGAGATAAAACCTCA AGTGTCCTCTT GCCTCCACTGATCACTCTCCTATGTTTATTTCCTCAAA

Canine exon 1 (leader) from LOC475754 (SEQ ID NO. 424) :

atgaggttcccttctcagctcctggggctgctgatgctctggatcc

Canine intron 1 from LOC475754 (SEQ ID NO. 425)

Caggtaaggacagggcggagatgaggaaagacatgggggcgtggatggtgagctcccctg gtgctgtttct ctccctgtgtattctgtgcatgggacagattgccctccaacagggggaatttaattttta gactgtgagaa ttaagaagaatataaaatatttgatgaacagtactttagtgagatgctaaagaagaaaga agtcactctgt cttgctatcttgggttttccatgataattgaatagatttaaaatataaatcaaaatcaaa atatgatttag cctaaaatatacaaaacccaaaatgattgaaatgtcttatactgtttctaacacaacttg tacttatctct cattattttaggatccagtggg

Canine 5' part of exon 2 (leader) from LOC475754 (SEQ ID NO. 426) aggatccagtggg

Canine VK from LOC475754 (SEQ ID NO. 427)

Gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctcc atctcctgcaa ggccagtcagagcctcctgcacagtgatggaaacacgtatttgaactggttccgacagaa gccaggccagt ctccacagcgtttaatctataaggtctccaacagagaccctggggtcccagacaggttca gtggcagcggg tcagggacagatttcaccctgagaatcagcagagtggaggctgacgatactggagtttat tactgcgggca aggtatacaagat

Mouse RSS heptamer (SEQ ID NO: 428)

CACAGTG Mouse sequence downstream of RSS heptamer (SEQ ID NO. 429)

ATACAGACTCTATCAAAAACTTCCTTGCCTGGGGCAGCCCAGCTGACAATGTGCAAT CTGAAGAGGAGCAG

AGAGCATCTTGTGTCTGTGTGAGAAGGAGGGGCTGGGATACATGAGTAATTCTTTGC AGCTGTGAGCTCTG

Igk version B

Sequence upstream of mouse Igkv 1-133 (SEQ ID NO. 430)

GCATTGAATAAACCAGTATAAACAAGCAAGCAAAGATAGATAGATAGATAGATAGATAGA TAGATAGATAC ATAGATAGATAGATAGATAGATAGATGATAGATAGATAGATAGATAGATAGATTTTTACG TATAATACAAT AAAAACATTCATTGTCCCTCTATTGGTGACTACTCAAGGAAAAAAATGTTCATATGCAAG AAAAAATGTTA TCATTACCAGATGATCCAGCAATCTAGCAATATATATATTGTTTATTCACAAAACATGAA TGAACCTTTTA AGAAGCTGTTACAGTGTAAAAATTAAGTTAAATCACTGAAGAACATATACTGTGTGATTT CATTCAAATGA AATTTGAGAAGTAAATATATATGTATATATATATATATGTAAAAAATATAAGTCTGAACT ACAAAAATTCA ATTTGTTTGATATGTAAGAATAAGAAAAATTGACCCCCAAAATTTGTTAATAATTAGGTA TGTGTATTTTT ATGAATATATAAGTATAATAATGCTTATAGTATACACTATTCTGAATCACATTTATTCCC TAAGTGTGTTC CCTTGATTATAATTAAAAGTATATTTTTTAAATACAGAGTCAGAGTACAGTCAATAAGGC GAAAATATAGT TGAATGATTTGCTTCAGCTTTTGTAATGTACTAGAGATTGTGAGTACAAAGTCTCAGAGC TCATTTTATCC CTGACAATAACCAGCTCTGTGCTTCAAGTACATTTCCATCTTTCTCTGAAATTTAGTCTT ATATAGATAGA CAAAATTTAAGTAAATTTCAAACTACACAGAACAACTAAGTTGTTGTTTCATATTGATAA TGGATTTGAAC TGCATTAACAGAACTTTAACATCCTGCTTATTCTCCCTTCAGCCATCATATTTTGCTTTA TTATTTTCACT TTTTGAGTTATTTTTCACATTCAGAAAGCTCACATAATTGTCACTTCTTTGTATACTGGT ATACAGACCAG AACATTTGCATATTGTTCCCTGGGGAGGTCTTTGCCCTGTTGGCCTGAGATAAAACCTCA AGTGTCCTCTT GCCTCCACTGATCACTCTCCTATGTTTATTTCCTCAAA

Mouse Igkv 1-133 exon 1 (leader) (SEQ ID NO. 431)

ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGG

Mouse Igkv 1-133 intron 1 (SEQ ID NO. 432)

GTAAGGAGTTTTGGAATGTGAGGGATGAGAATGGGGATGGAGGGTGATCTCTGGATGCCT ATGTGTGCTGT TTATTTGTGGTGGGGCAGGTCATATCTTCCAGAATGTGAGGTTTTGTTACATCCTAATGA GATATTCCACA TGGAACAGTATCTGTACTAAGATCAGTATTCTGACATAGATTGGATGGAGTGGTATAGAC TCCATCTATAA TGGATGATGTTTAGAAACTTCAACACTTGTTTTATGACAAAGCATTTGATATATAATATT TTTAAATCTGA AAAACTGCTAGGATCTTACTTGAAAGGAATAGCATAAAAGATTTCACAAAGGTTGCTCAG GATCTTTGCAC ATGATTTTCCACTATTGTATTGTAATTTCAG

Mouse Igkv 1-133 5' part of exon 2 (leader) (SEQ ID NO. 433)

AAACCAACGGT

Canine VK from LOC475754 (SEQ ID NO. 434)

Gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctcc atctcctgcaa ggccagtcagagcctcctgcacagtgatggaaacacgtatttgaactggttccgacagaa gccaggccagt ctccacagcgtttaatctataaggtctccaacagagaccctggggtcccagacaggttca gtggcagcggg tcagggacagatttcaccctgagaatcagcagagtggaggctgacgatactggagtttat tactgcgggca aggtatacaagat

Mouse RSS heptamer (SEQ ID NO: 435)

CACAGTG

Mouse sequence downstream of RSS heptamer (SEQ ID NO. 436)

ATACAGACTCTATCAAAAACTTCCTTGCCTGGGGCAGCCCAGCTGACAATGTGCAAT CTGAAGAGGAGCAG AGAGCATCTTGTGTCTGTGTGAGAAGGAGGGGCTGGGATACATGAGTAATTCTTTGCAGC TGTGAGCTCTG