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Title:
METHOD FOR TARGETED HOMOLOGOUS GENE REPLACEMENT IN KINETOPLASTIDAE
Document Type and Number:
WIPO Patent Application WO/2002/090556
Kind Code:
A1
Abstract:
This invention pertains to a method for creating homozygous gene replacements in cells resulting e.g. in complete loss of targeted gene function or over-expression of heterologous genes comprising transfecting said cells with at least one circular DNA construct comprising a selectable marker flanked by nucleotide sequences complementary to the 5' and 3' flanking regions of the genetic locus by said DNA sequences and selecting for the replacement of said genetic locus from the genome of said cells and if appropriate extracting said selectable marker out of the genome of the said cells.

Inventors:
ALEXANDROV KIRILL (DE)
BREITLING REINHARD (DE)
Application Number:
PCT/EP2002/005023
Publication Date:
November 14, 2002
Filing Date:
May 07, 2002
Export Citation:
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Assignee:
JENA BIOSCIENCE GMBH (DE)
ALEXANDROV KIRILL (DE)
BREITLING REINHARD (DE)
International Classes:
C07K14/44; C12N1/10; C12N15/10; C12N15/79; (IPC1-7): C12N15/79; C12N15/90
Domestic Patent References:
WO2000058483A22000-10-05
Foreign References:
EP0829540A11998-03-18
Other References:
DOS SANTOS W.G. & BUCK G.A.: "Simultaneous expression of neomycin phosphotranferase and green fluorescent protein genes in Trypanosoma cruzi", J. PARASITOL., vol. 86, no. 6, 2000, pages 1281 - 1288, XP001013424
EID J. & SOLLNER-WEBB B.: "Stable integrative transformation of trypanosoma brucei that occur exclusively by homologous recombination", PROC. NATL. ACAD. SCI. USA, vol. 88, March 1991 (1991-03-01), pages 2118 - 2121, XP002175992
CLAYTON C.E.: "Genetic manipulation in Kinetoplastida", PARASITOLOGY TODAY, vol. 15, no. 9, 1999, pages 372 - 378, XP002175993
HARIHARAN S. ET AL.: "Stable transformation of trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption", MOL. BIOCHEM. PARASITOL., vol. 57, 1993, pages 15 - 30, XP001013654
HWANG H.-Y. ET AL.: "Creation of homozygous mutants of Leishmania donovani with single targeting constructs", J. BIOL. CHEM., vol. 271, no. 48, 29 November 1996 (1996-11-29), pages 30840 - 30846, XP002175994
CRUZ A. ET AL.: "Double targeted gene replacement for creating null mutants", PROC. NATL. ACAD. SCI. USA, vol. 88, August 1991 (1991-08-01), pages 7170 - 7174, XP002052132
BROOKS D.R. ET AL.: "Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase", FEMS MICROBIOLOGY LETTERS, vol. 186, 2000, pages 287 - 291, XP001013609
ZHANG W.W. ET AL.: "The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins", NUCL. ACIDS RES., vol. 23, no. 20, 1995, pages 4073 - 4080, XP002147127
LEE M.G. & VAN DER PLOEG L.H.T.: "The hygromycin B-resistance-encoding gene as a selectable marker for stable transformation of Trypanosoma brucei", GENE, vol. 105, 1991, pages 255 - 257, XP001019015
Attorney, Agent or Firm:
Ackermann, Joachim (Frankfurt, DE)
Download PDF:
Claims:
Claims : 201jb0
1. wo 1. A method for producing genetic transformed cells by homologous recombination, wherein said cells are: a) transfected with at least one circular DNA construct comprising selectable marker flanked by nucleotide sequences complementary to the 5'and 3'flanking regions of an actively transcribed genetic locus to be replaced, b) replacement of said genetic locus by the DNA sequence comprising the selectable marker by homologous recombination under increasing drug pressure and c) selecting for the replacement of said genetic locus from the genome of said cells.
2. A method of claim 1 wherein said transfecting circular DNA construct comprising at least two repeat regions, preferably perfect repeats, flanking said selection marker and wherein the repeat regions and said selection marker is flanked by nucleotide sequences complementary to the 5'and 3' flanking regions of the genetic locus to be replaced and wherein subsequent to the replacement (step b)) and selection (step c)) the selection marker is removed out of the genome of the cells by homologous recombination mediated by the repeat regions.
3. A method of claim 1 or claim 2 wherein said circular DNA construct comprising a gene of interest.
4. A method of claim 1 wherein the cells comprising at least one genomic selection marker integrated by homologous recombination replacement according to claim 1 are subsequently transfected with a circular DNA construct comprising a gene of interest flanked by nucleotide sequences complementary to the 5'and 3'flanking regions of the actively transcribed selection marker and replacement of said selection marker by said gene of interest by homologous recombination.
5. A method as claimed in any of the precedingclaims wherein the circular DNA construct comprises an additional reporter gene between the 5'and 3'flanking regions.
6. A method as claimed in claim 5 wherein the reporter gene is fused to the selection marker gene.
7. A method as claimed in claim 5 or claim 6 wherein the reporter gene is the GFP gene, luciferase gene, ßgalactosidase or thymidine kinase gene.
8. A method as claimed in any of the preceding claims wherein said 5'and 3' DNA sequences are actively transcribed regions of 18S, 28S or 5S gene or of intergenic regions in between them.
9. A method as claimed in claim 3 or claim 4 wherein said gene of interest is flanked by DNA sequences of an actively transcribed gene of the transfected cells.
10. A method claimed in claim 6 wherein said DNA sequences are actively transcribed regions of the Kinetoplastida a, 3tubulin, calmodulin or gp63 gene.
11. A method as claimed in any of the preceding claims wherein the selection marker is an antibiotic resistant gene or an auxotrophic marker.
12. A method of claim 1 or 2 wherein said genetic locus to be replaced comprises a gene.
13. A method of claim 1 or 2 wherein said genetic locus comprises one, two or multiple alleles.
14. A method of claim 1 or 2 wherein said genetic locus to be replaced contains protease gene, a glycosyl transferase gene or an metabolic enzyme gene or a structural protein.
15. A method of claim 1 or 2 wherein the replacement of said genetic locus leads to conditional auxotrophy.
16. A method as claimed in any of the preceding claims wherein the genetic locus to be replaced is a strong transcribed locus and/or the selection marker in the circular DNA construct is weak or conditionally transcribable.
17. A method as claimed in any of the preceding claims wherein the cells are transfected with a mixture of circular DNA constructs different constructs are flanked by nucleotide sequences complementary to the 5'and 3'flanking regions of different actively transcribed genetic loci to be replaced.
18. A method as claimed in any of claims 1 to 16 wherein the cells are sequentially transfected with different circular DNA constructs and each construct is flanked by nucleotide sequences complementary to the 5'and 3' flanking regions of a different actively transcribed genetic locus to be replaced and before the second and all further transfection steps the selection marker is removed.
19. A method as claimed in claim 17 or claim 18 wherein only one selection marker gene is used for multiple replacements generated with a method of claim 1 or 2.
20. A method of claim 1 or 2 wherein the extraction of said selectable marker out of the genome of said cells is carried out using conditions promoting deletion of said selection marker.
21. A method of claim 1 or 2 wherein the extraction of said selectable marker out of the genome of said cells is carried out using conditions promoting homologous recombination.
22. A method as claimed in any of the preceding claims wherein the replacement by homologous recombination leads to homozygous transfected cells.
23. A method as claimed in any of the preceding claims wherein the increasing drug pressure is generated by increasing amounts of said drug, preferably antibiotics, in the medium or by specific inhibition of the transcription of the selection marker gene of the circular DNA construct.
24. A method as claimed in any of the preceding claims wherein said transfected cells are unicellular organisms.
25. A method as claimed in any of the preceding claims wherein said transfected cells are diploide or multiploide.
26. A method as claimed in any of the preceding claims wherein said transfected cells are Kinetoplastidae.
27. A method as claimed in any of the preceding claims wherein said Kinetoplastidae is a parasitic protozoa.
28. A method as claimed in any of the preceding claims wherein said parasitic protozoa is a member of the genus Leishmania or Trypanosomatidae.
29. A genetic mutant of a Kinetopalstidae organism produced according to the method as claimed in any of the preceding claims.
30. Kinetopalstidea as claimed in the previous claim wherein the genome of said Kinetopalstidae is devoid of a naturallyoccurring genetic locus that modulates virulence of said organism in a host.
31. Kinetopalstidae of claim 29 wherein said genetic locus modulates persistence of said organism in said host in vivo and is not required for growth of said organism in vitro.
32. Kinetopalstidae wherein the genome of said Kinetopalstidae is devoid of a naturallyoccurring genetic locus encoding a protease, glycosyl transferase or an metabolic enzyme or a structural protein.
33. A circular vector or plasmid comprising a selectable marker, preferably weak or conditionally transcribable, flanked by nucleotide sequences complementary to the 5'and 3'flanking regions of an actively transcribed genetic locus to be replaced.
34. A circular vector or plasmid as claimed in claim 33 comprising further a gene of interest or a gene of interest flanked by regions of an actively transcribed host gene and/or a reporter gene and/or repeat regions, preferably perfect repeats, flanking the selection marker or the reporter gene or the fused selection marker/reporter gene.
Description:
METHOD FOR TARGETED HOMOLOGOUS GENE REPLACEMENT IN KINETOPLASTIDAE Description This invention relates to methods of creating targeted homologous DNA replacements in cells resulting in complete loss of targeted gene function (i. e. deletion) or over-expression of desired genes of interest (e. g. heterologous genes).

Members of the Kinetoplastidae group are archaic unicellular organisms (Stevens, J.

R. et al. (1999)). Kinetoplastidae are diploid at most loci and appear to be exclusively asexual. Genetic manipulation of Kinetoplastidae often requires deletion or disruption of both alleles of the gene or alternatively insertion of a new gene (Clayton, C. E.

(1999)). This is normally achieved by a homologous recombination of the endogenous gene with the construct that comprises DNA encoding the selectable marker flanked by DNA complementary to flanking regions of the gene. In the state of the art, disruption of both alleles is achieved by using two independent selectable markers (Cruz, A. et al. (1991)). This procedure results in strains resistant to antibiotics and render inapplicable when several genes have to be deleted due to the limited number of available antibiotic resistance genes, precluding usage of such markers in further genetic engineering. Moreover, selection on several antibiotics has negative influence on the growth rates. The potential ability to delete multiple genes is particularly important since genes are present in multiple copies in the genome, as given in Kinetoplastidae genome, wherein multiple copies are often arranged in tandems. It is known that such gene clusters can be deleted by UV-or chemical mutagenesis. This has the disadvantage of creating secondary mutations as well as the low predictability of the outcome. Homologous recombination is also employed when a copy of a gene needs to be inserted into the Kinetoplastidae genome (Eid, J.

E. et al. (1991); Blundell, P. A. et al. (1996)). In this case a construct composing a split sequence of a homologous gene containing inside the gene of interest is flanked with untranslated regions (UTR) and operably linked to a selectable marker (Misslitz A.). Similar to the methods the number of integrations achieved using this method is limited by the number of available antibiotic resistance genes (Clayton, C. E. (1999)).

Therefore uncomplicated methods that allow insertion of multiple copies of the same gene in order to achieve high-level overexpression are needed. It is also highly desired to develop a method that allows co-expression of multiple heterologous genes. It would be also advantageous to make available a method of removing the antibiotic resistance gene out of the genome of the Kinetoplastidae once the gene of interest is integrated.

In a further aspect the invention relates to a method of providing homologous gene replacements resulting in loss of the targeted DNA sequence in cells by carrying out homologous gene replacements with circular DNA constructs.

The circular construct comprises DNA encoding a selectable marker, preferably flanked by DNA complementary to flanking regions of the genetic locus to be replaced. The cell is transfected with the circular construct, which maintained episomally and upon drug pressure the selection of cells can be carried out wherein the circular construct is recombined with the genomic copy and replace the targeted genes within at least one genetic locus in at least one allele. The transfected cells are selected by expression of the selectable marker, which indicates homologous replacement of the gene. The selection for double replacement is carried out through quantification of the drug resistance or/and quantification of the expression level, using an operably linked reporter gene.

As drugs for the selection of recombined cells can be used all compounds which discriminate cells with episomally maintained circular constructs, such as antibiotics or inhibitors of the transcription of the selection marker gene located on the circular construct. The presence of said drugs which leads to the selection of the recombined cells.

The method comprises targeting of at least one allele-in a preferred embodiment of all alleles-of the genetic locus to be replaced with DNA constructs bearing single selectable markers.

The circular DNA constructs can comprise additionally a heterologous nucleic acid sequence coding for a protein of interest operably linked to a resistance marker gene flanked by segments of an actively transcribed gene of the host cell.

After the homologous replacement and the selection for said replacement is carried out, the selection marker can be removed.

It will be understood that throughout the specification and claims the use of the term genetic locus means any genetic material, in particular genes, alleles, which is capable of recombination.

Therefore one embodiment of the invention is a method for producing genetic transformed cells by homologous recombination, wherein said cells are: a) transfected with at least one circular DNA construct comprising selectable marker flanked by nucleotide sequences complementary to the 5'and 3' flanking regions of an actively transcribed genetic locus to be replaced, b) replacement of said genetic locus by the DNA sequence comprising the selectable marker by homologous recombination under drug pressure and c) selecting for the replacement of said genetic locus from the genome of said cells and if appropriate d) subsequent removal of said selectable marker out of the genome of said cells.

The drug pressure leads to conditions promoting the replacement and selection of the genetic locus to be replaced by the DNA sequence between the flanking regions complementary to the flanking regions of said genetic locus. The transcription of the selection marker gene of the circular DNA construct leads to maintenance of episomally transfected cells in the presence of drug pressure. Under the stringent drug pressure only cells are maintained wherein the homologous replacement of the envisaged genetic locus by the DNA fragment comprising the selection marker gene has taken place. Preferably the genetic locus to be replaced is a strong transcribed locus and the selection marker in the circular DNA construct is weak or conditionally transcribable. Increasing drug pressure can be generated e. g. by increasing amounts of said drug, in particular of antibiotics, in the medium or by specific inhibition of the transcription of the selection marker gene of the circular DNA construct (see Clayton C. 1999).

The method can be used e. g. in Kinetoplastidae with the purpose of providing mutations in different genes. Such mutations can be used for studies of these organisms or for providing organisms with useful characteristics such as auxotrophic mutants, protease deficient strains for recombinant protein production, strains with disrupted metabolic pathways for metabolic research purposes or other bioengineering purposes or construction of the attenuated strains for use as living vaccines.

Therefore, the genetic locus to be replaced can contain one or more genes, one or more alleles, in particular multiple alleles. Preferably, such genetic loci, genes or alleles to be replaced in the cell and/or organism, preferably in Kinetoplastidae, comprises, but is not limited to, a genetic locus that modulates virulences and persistences, genes encoding a protease, glycosyl transferase or an metabolic enzyme or structural proteins.

Organisms of the order Kinetoplastidae have a unique organelle so called the kinetoplast, an appendix of their single mitochondrion located near the basal body of the flagellum that contains a network of thousands of small interlocking circular DNAs. Kinetoplastidae have a size of 50-400 micron. Kinetoplastidae are among the most ancient eukaryotes, with rRNA lineages extending further back than those of animals, plants, and even fungi (Beverley, 1996). A lot of representatives of Kinetoplastidae were isolated and maintained in culture for an extended period of times. Cultivation and fermentation could be carried out on both, completely or partially defined media (Melo, 1981). Kinetoplastidae could be cultivated and fermented on both, liquid and solid media. Grown in the liquid media Kinetoplastidae form suspensions whereas plating on solid media leads to formation of colonies (Hill and Fahey, 1987).

It will be understood that throughout the specification and claims the use of the term "Kinetoplastidae"refers not only to organisms/hosts encompassed in the aforementioned species, but also includes those species in alternate classification schemes, but which possess the same morphological and cultural characteristics or features defined above, and may be synonyms of"Kinetoplastidae" According to another embodiment of the invention the circular construct comprises a DNA encoding expression cassette that includes a gene of interest operably linked to a selectable marker flanked by DNA complementary to flanking regions of the actively transcribed gene such as but not limited to genes of the ribosomal gene cluster such as 18S, 28S, 5S or intergenic regions in between them. The cells are transfected with the circular construct or a mixture of constructs which maintained episomally and following selection procedure by placing the expression cassette into the actively transcribed genetic locus by recombination. The transfected organisms are selected for expression of the selectable marker, which indicates integration of the gene cassette. For integration of the multiple genes this procedure can be performed either parallel with a mixture of several constructs or sequentially.

Modifying the UTR of the marker gene and/or varying the selection pressure can modulate the number of integrated constructs. This procedure can be combined with several selectable markers. The selection for genomic integration is carried out through quantification of the drug resistance or/and quantification of the expression level, using an operably linked marker reporter gene.

Furthermore, it is an advantage of the present invention, that the method allows multiple circles of the steps a.)-d.) (page 3 and see Fig 1). This depends on the fact that the removal of the selectable marker does not lead to its exhaustion. Therefore, it is advantegeous that only few selectable markers are utilised.

The method of the present invention can be preferably used in Kinetoplastidae and other diploid unicellular organisms with purpose of providing strains for the over- expression of a single gene achieved by integration of multiple copies. Further this invention preferably relates to construction of Kinetoplastidae strains or strains of other diploid unicellular organisms for over-expressing several genes simultaneously.

Such mutation can be used for studies of these organisms or for providing organisms with useful characteristics such as for instance bearing novel metabolic or glycosylation pathways, signal transduction cascades etc. These strains can be used to overproduce a specific protein or group of proteins such as multi-subunital enzymes. Such strains can be used for in vitro drug screening.

In another embodiment of the present invention the integration construct contains an inducible gene expression cassette that is inserted in the reverse orientation to the selection marker. Such cassette can contain the gene of interest prefaced with a promoter gene such as rRNA promoter or promoter for a foreign polymerase such as for instance but not limited to the promoters for T7, T3 or a RNA polymerase I from a different species for instance another Kinetoplastidae. Such promoter can contain an operator sequence such as but not limited to TET or Lac. This construct is transformed into a Kinetoplastidae species bearing a gene for foreign RNA polymerase. This assembly can ensure that the gene of interest is silent until inductor such for instance tetracycline is added. In the same time integration can take place due to the read through transcription of the antibiotic resistance gene.

As a gene of interest can be used and targeted to express enzymes such as catalase, laccase, phenoloxidase, oxidase, oxidoreductases, cellulase xylanase, peroxidase, lipase, hydrolase, esterase, cutinase, protease and other proteolytic enzymes, aminopeptidase, carboxypeptidase, phytase, lyase, pectinase and other pectinolytic enzymes, amylase glucoamylase, a-galactosidase, ß-galactosidase, a- glucosidase, ß-glucosidase, mannosidase, isomerase, invertase, transferase, ribonuclease, chitinase, and deoxyribonuclease. It will be understood by those skilled in the art that the term"enzymes"includes not only native enzymes, but also those enzymes which have been modified by amino acid substitutions, deletions, additions, or other modifications which may be made to enhance activity, thermostability, pH tolerance and the like.

According to another preferred embodiment homozygous gene replacement can be achieved in cells e. g. in Kinetoplastidae or other unicellular diploid organisms by targeting the alleles of a gene with a single circular genetic construct containing single selectable marker. The gene coding for the selectable marker can be later removed by induced homologous recombination. The method can be in particular used to study gene function e. g. in Kinetoplastidae, create strains over-expressing heterologous proteins, protein complexes or parts of functional biological pathways, create strains deficient in intra and extra-cellular proteases and to create attenuated strains of parasitic Kinetoplastidae used as living vaccines.

This embodiment pertains further to a method of constructing transgenic cells using the technical teach described above followed by targeted removal of the antibiotic resistance genes from the genome of the cell. The construct comprises DNA encoding a selectable marker or a fusion protein of the selectable marker and conditionally toxic gene or/and reporter gene forming a selection cassette. The untranslated sequences derived from an actively transcribed gene or an artificial sequence are added in duplicate on the 3'and 5'end forming a repeat region, preferably a perfect repeat. This cassette is flanked by targeting region of DNA complementary to flanking regions of the gene, or portion thereof, or an untranslated region of the genome. Upon genomic integration mediated by selection with the said selectable marker, the marker gene can be deleted by homologous recombination. In case of fusion proteins between selectable marker and conditionally toxic protein deletion of the resistance marker can be stimulated by treatment with agents that confer cyto-toxicity to the conditionally toxic protein. The loss of marker is established by measuring the activity of the selection marker gene or of the associated reporter gene.

This method can be used in particular for the transformation of diploid or multiploide cells or organisms e. g. for the transformation of Kinetoplastidae with purpose of creating strains expressing multiple heterologous genes or having multiple genes deleted. Such strains can be liberated from the antibiotic resistance genes allowing to perform further rounds of genetic modification.

Furthermore the present invention provides methods for generating homozygous gene replacements that can be created by targeting two or more alleles of the gene to be replaced with DNA constructs containing a single selectable marker such as, but not limited to, antibiotic resistance genes or auxotrophic markers. According to the preferred embodiment of this invention, the cells are transfected with a circular DNA construct that comprises DNA encoding selectable marker flanked by DNA complementary to 5'and 3'flanking regions of one allele of the gene, or portion thereof, to be replaced. The cells are transfected with the circular construct that is maintained episomaly and subjected to increasing selection pressure resulting in the integration of the constructs into each allele in place of the targeted gene. The transfected cells are selected for expression of the selectable marker, which indicates homozygous replacement of the gene : The presence of an endogenous gene disrupted at both alleles in a diploid cell is conferred by Southern blotting.

In another preferred embodiment the selection marker is fused to a reporter gene such as for example Green Fluorescent Protein, luciferase,-galactosidase etc. In such configuration the number of genome integrated copies can be measured based on the activity of the fused reporter gene. The reference strain is constructed by that has been transforming the organisms with the linear version of the integration construct ensuring a single genomic integration (Misslitz A.). Comparing the fluorescence of clones obtained through transformation with the circular construct with clones obtained with linearized construct one can establish the number of copies integrated e. g. in the Kinetoplastidae genome.

The DNA constructs for targeted replacement are constructed by standard techniques of gene targeting methodology (see Sambrook, J. et al. supra). In a preferred embodiment, nucleic acids or nucleotide sequences according to the invention are DNA or RNA, preferably a double-stranded DNA. Typically, constructs are circular DNA fragments that are assembled in a DNA vector, such as a plasmid.

However, other suitable or appropriate nucleic acids constructs are not excluded from the present invention, and shall be hereby incorporated, in particular, those which are able to recombine within a genetic locus.

The skilled person in the art will also recognize that the successful transformation of the host cell described herein is not limited to the use of the vectors, plasmids and selection markers specifically exemplified. Generally speaking, those techniques are implicated, which are useful with the transfected cells of the present invention.

The method can be used to replace a complete gene, a portion of a gene or gene clusters. In order to construct the targeting vector the flanking regions of the gene to be replaced are isolated or synthesized. Generally, sequences consisting of at least 60bp are needed for efficient recombination. The selectable marker is rendered operable by supplying it with 5'and 3'untranslated regions of an efficiently expressed gene. This assembly is further referred as a"selectable marker unit". The selectable marker unit is flanked by the portions of the genes to be disrupted and combined with a bacterial origin of replication and bacterial antibiotic resistance gene such as but not limited to beta-lactomase.

Such selectable marker units used in the present invention contain typically markers, which confer drug resistance. That includes but is not limited to hygromycin phosphotransferase (HYG) gene in combination with hygromycin, neomycin phosphotransferase (NEO) gene in combination with G418, product of the Streptoalloteichus hindustanus BLE gene (BLE) in combination with phleomycin and streptothricin acetyltransferase encoding (SAT) gene in combination with nourseothricin that are used for selection of recombinant clones. The selection marker can be also a gene complementing an auxotrophic mutation such as for instance an ornithine decarboxylase (ODC) (GeneBank accession number AF159564) in combination with an ODC null mutant strain.

The method of this invention can be used to provide strains of Kinetoplastidae with attenuated virulence for use in live vaccines. This method can be also used for providing auxotrophic mutants of Kinetoplastidae that can be used for further genetic engineering. This method can be also used for creating strains deficient in proteases or other enzymes and thus be suitable for production of recombinant proteins and other compounds.

The invention is illustrated further by the following exemplification of the integration of multiple copies of EFGP protein into the small subunit of the rRNA cluster in Leishmania tarentolae (Example 1).

The present invention also provides methods for integration of multiple genes (either identical or diverse) into the actively transcribed regions of the Kinetoplastidae genome thus achieving over-expression of the said genes. According to a preferred embodiment of this invention, the vector system preferably comprises circular nucleic acid and heterologous nucleic acid coding for a protein of interest flanked by intergenic regions of an actively translated host protein operably linked to a resistance marker gene flanked by segments of an actively transcribed Kinetoplastidae gene. Such genes include but are not limited to subunits of ribosomal RNA genes, a or p tubulin genes, calmodulin gene, gp63 gene. The integration procedure is performed as described above. The number of integrated gene can be influenced by increasing the selection pressure and by modifying the 5'UTR of the selectable marker. Substitutions, deletions, or replacements of the homologous 3' or/and 5'UTR or its parts with random or heterologous sequences leads to a less efficient RNA processing and translation of the selectable marker gene and thus increases the number of the episomal copies as well as number of chromosomal integrations. Combination of these two parameters can be used to obtain desired number of integrated copies and level of protein expression.

The present invention also provides methods for removal of selection marker genes integrated into the Kinetoplastidae genome. According to this method the integration constructs of the above-described type are supplied with repeating regions before and after the marker genes. The repeated region can comprise either an identical intergenic region placed before and after the selection marker or a synthetic sequence that does not intervene with the RNA maturation. The construct is integrated into the genome by homologous recombination as described above and upon integration the selection pressure is removed and the transgenic organisms are cultivated for several generation. The selection marker is excised by homologous recombination and its absence is established by the emergence of sensitivity to the selection agent such as antibiotics or loss of the heterotrophic phenotype. This approach allows for multiple rounds of integration of the selection marker as well as operably associated gene. Moreover, this procedure allows sequential disruption of both alleles resulting in the homozygous replacement of the gene and subsequent reversal of the selection marker bearing phenotype. This procedure also allows integration of multiple functional genes and removal of the associated antibiotics and antibiotic resistant genes used for their integration (see Fig. 1).

The invention is illustrated further by the following exemplification of the integration of EFGP-antibiotic fusion gene into the rRNA cluster of Leishmania tarentolae and its subsequent removal resulting in emergence of the drug sensitive organisms.

(Example 2) In another modification of a preferred invention the selectable marker is fused with a reporter gene such as GFP. This allows the selection of the clones with excised selection marker by loss of the transformation associated fluorescence.

In another modification of the preferred invention the selectable marker can be fused to the conditionally toxic gene. Such gene can be rendered toxic to the cell by addition of exogenous compound. The clones that lost the selection marker cassette can be grown on the medium containing the exogenous compound.

The present invention relates further to recombinant vectors and strategies for their application which allow gene deletion or insertion into the genome of cells, in particular of unicellular diploid organisms like Kinetoplastidae for the construction of strains with new properties such overproduction of proteins or small molecules, altered sensitivity to specific chemical compounds.

Brief description of the figures: FIG. 1: Overview of a preferred embodiment including the removal of chromosomal integrated selection marker genes. The integration of the expression cassette is carried out as described in Example 2. The expression cassette possesses additionally repeating regions before and after the marker gene. After the selection of cells with a marker gene integrated in the chromosomal locus the marker gene itself can be removed by recombination out of the chromosomal locus. After removal of the marker gene a second transfection can be carried out with the same marker gene.

FIG 2: Integration plasmid pF4egfp1. 4sat1 used for integration of EGFP gene expression cassette into SSU locus. Abbreviations: 5'ssu-5'part of 18S rRNA gene of L. tarentolae Acc# gi159399 (nt 123-842) modif, 3'ssu-3'part of 18S rRNA gene of L. tarentolae Acc# gi159399 (nt 1055-8422139), 0.4k IR camBA-intergenic region from calmodulin operon of L. tarentolae (descr. here), 1.4k IR camCB-intergenic region from calmodulin operon of L. tarentolae (descr. here), 1.7k-IR- intergenic region from DHFR-TS gene Acc# X51734 (nt. 6- 573) of L. major, sat-nourseothricin resistance gene Acc# M63169 (nt. 106-632), egfp-green fluorescence gene Acc# U55762 (nt. 677-1407) FIG 3 : Fig. 2 EGFP expression levels (determined by fluorescence of the suspension cultures) of recombinant L. tarentolae strains obtained by electroporation with either circular (lans 1-11) or linearized (lanes 12-22). The height of the bars represents the arbitrary fluorescence units. Chromosomal integration was confirmed by genomic PCR and Southern blot analysis with the EGFP gene as the probe.

FIG 4: Schematic representation of pF4blegfp1. 4sat1. 4 plasmid containing two selection markers that was used for analysis of reversibility of selection marker integration. The black triangles denote the repetitive parts derived from the camCB- intergenic region of calmodulin operon of L. tarentolae The following examples serve to describe the invention in greater detail, but without restricting the same to the products and embodiments described in the examples.

EXAMPLE 1 Expression of EGFP target protein in Leishmania tarentolae by chromosomal integration of circular expression plasmid pF4egfp1. 4sat1.

Construction of expression plasmid pF4egfp1. 4sat1.

The EGFP expression plasmid pF4egfp1. 4sat1 (Fig. 2) was generated by insertion of Ncol x Notl trimmed egfp cassette obtained from pEGFP-N1 (Acc# U55762 nt. 677- 1407, Clontech Labs. Inc.) into plasmid pJBS1.4sat opened Ncol x Notl. Construction of pJBS1.4sat prototype plasmid was described previously (Breitling et al. 2002). The ccc form of plasmid DNA was isolated from recombinant E. coli strain and column purified. For a control experiment, a fraction of 5 ug of plasmid DNA was linearized with restriction enzyme Swal.

Transfection of Leishmania tarentolae with expression plasmid pF4egfp1. 4sat1.

Submerse cultures of Leishmania tarentolae were grown in BHI (Difco) supplemented with 5 ug/ml hemin to a cell density of approx. 1 OD at 600 nm. Per transfection 1 mi culture was washed and concentrated in 0.4 mi electroporation buffer (approx. 4 x 107 cells/0. 4 ml). Electroporation was performed with Bio-Rad Gene Pulser II as described (Beverley & Clayon, 1993; Meth. Mol. Biol. Vol 21 p.

333-347). In parallel, 5 pg of circular or linearized with Swal plasmid pF4egfp1. 4sat1 were electroporated into L. tarentolae cells. Recombinant clones were selected on solidified M199 media supplemented with 50 lug/ml nourseothricin and 5 lug/ml hemin.

Colonies were grown as submerse cultures in BHI supplemented with 100 ug/ml nourseothricin and 5 ug/ml hemin and analysed for EGFP production and genetic structure.

Analysis of EGFP expression and genetic structure of recombinant L. tarentolae transfected with pF4egfp1. 4sat1.

Fluorimetric assays of EGFP expression were performed with 0.2 ml samples of normalized cell lysates (10mM Tris pH 8.0,1% Triton) in 96 well format on FLUOstar Galaxy (BMG Labtechnologies) at E485 nm excitation and E520 nm emission. In Western blots of cell extracts EGFP was detected with rabbit anti-GFP antibodies.

Fig. 3 shows EGFP expression levels of recombinant L. tarentolae strains obtained by electroporation with either circular (lans 1-11) or linearized (lanes 12-22) pF4egfp1.4sat1.

Whereas the clones obtained with linearized plasmid showed homogeneous EGFP expression levels, the clones obtained with circular plasmid displayed differential expression levels. Particularly, clone 10 expressed EGFP at 1.8 times higher level as compared to linear integrants. The genetic analysis of these clones was performed by diagnostic PCR of genomic DNA with specific primer pairs. Correct genomic integration of linearized plasmids into the SSU locus was demonstrated by a characteristic 2.3 kb fragment, obtained with forward sat primer F2999 (CCTAGTATGAAGATTTCGGTGATC) and reverse ssu primer F3002 (CTGCAGGTTCACCTACAGCTAC) hybridizing outside of the plasmid derived sequence within the chromosomal 3'ssu region. Additional evidence was obtained from diagnostic PCR with egfp gene specific forward primer A2241 (GCC ATG GTG AGC AAG GGC) and reverse ssu primer F3002, generating a characteristic 4.4 kb fragment.

The evidence was obtained, that in clone 10 the circularly transfected plasmid also was integrated into the chromosomal ssu locus. This evidence was obtained from the characteristic 2.3 kb fragment, generated with forward sat primer F2999 and reverse ssu primer F3002. This pattern can be explained by homologous recombination of one of the ssu regions present on the plasmid (in this case the 3'ssu part) with a chromosomal ssu copy by a Campbell-like integration process (single crossover).

Since the expression level of clone 10 was almost two times higher than of the linear integrants, obviously, multiple copies of the plasmid can integrate by this mechanism.

EXAMPLE 2 Deletion of chromosomal markers from recombinant Leishmania tarentolae following integration of expression plasmid pF4blegfp1. 4sat1. 4.

Construction of dual marker plasmid pF4blegfp1. 4sat1. 4 The dual marker plasmid pF4blegfp1. 4sat1. 4 (Fig. 4) was generated by a two step procedure from plasmid pF4egfp1. 4sat (Fig. 2). In the first step the intermediate plasmid pF4blegfp1. 4sat was obtained by insertion of Ncol trimmed ble cassette with the ORF for phleomycin-bleomycin binding protein of Streptoalloteichus hindustanus (Acc# X52869 nt 2-382) conferring bleomycin/phleomycin/zeocin resistance obtained with primers A708 (TAA TAA GGA TCC ACC GCA TGG CCA AGT TGA CCA GTG) and A880 (TAA TAA CCA TGG CAG ATC TGT CCT GCT CCT CGG CCA C) from pLEW82 (Wirtz et al., 1999) into plasmid pF4egfp1. 4sat (Fig. 2) opened Ncol generating an in frame fusion of ble and egfp ORFs. In the second step the 3'UTR of sat marker gene of pF4blegfp1. 4sat was replaced by a second copy of the 1.4k IR from calmodulin operon of L. tarentolae as Spel x Fsel fragment. obtained with primers A1501 (CTA CTA ACT AGT CCT CCT CCT CCT TTC TTG TTC CTT) and A1502 (TAA TAA GGC CGG CCG GCT GCT GTG GAG GTG TGT AG).

Transfection of Leishmania tarentolae with dual marker plasmid pF4blegfp1. 4sat1. 4.

Leishmania tarentolae was transfected with 5 ug of Swal linearized plasmid pF4blegfp1. 4sat1. 4 as described in EXAMPLE1. Recombinant clones were selected with nourseothricin. Colonies were grown as submerse cultures in BHI supplemented with 100 pg/ml nourseothricin and 5 ug/ml hemin and analysed for EGFP production and genetic structure as described in EXAMPLE1. Correct genomic integration of linearized plasmids into the ssu locus was demonstrated by diagnostic PCR. The functionality of the Ble-EGFP fusion protein encoded by the blegfp fusion gene was demonstrated by zeocin resistance of the clones and EGFP expression. In comparison to EGFP the Ble-EGFP fusion protein displayed approx. 50% of the fluorescence. Western blots of cell extracts with rabbit anti-GFP antibodies demonstrated the same expression levels of both of the proteins in recombinant L. tarentolae strains.

Deletion of chromosomal markers from recombinant Leishmania tarentolae.

Since both of the marker genes of the F4blegfp1. 4sat1. 4 region were flanked by 110 bp repetitive sequences, this construct was suitable for an approach to delete one of the markers from the chromosome by homologous recombination. To this end the recombinant L. tarentolae strain harbouring the F4blegfp1. 4sat1. 4 region in the ssu locus was passaged for 10 rounds by 1: 50 dilutions in submerse cultures under three different conditions in parallel : a) without selection, b) with zeocin (ZEO) selection, c) with nourseothricin (NTC) selection. Following this cultivation colonies were obtained on solidified media and grown in submerse cultures. Ten clones of each variant were analysed for antibiotic resistance and EGFP expression. The results are shown in Tab. 1.

Tab. 1 SelectionPhenotype genotype a) NO 50% NTCR, ZeoS, EGFP-sat+, blegfp- 50% NTCS, ZeoS, EGFP-sat-, blegfp- b) ZEO 100% NTCR, ZeoR, EGFP+ sat+, blegfp+ c) NTC 100% NTCR, ZeoS, EGFP-sat+, blegfp- The genomic analysis of these clones by diagnostic PCR with gene specific primer pairs confirmed the phenotypes observed.

These results demonstrated, that chromosomal markers may be deleted from the chromosome by homologous recombination. In variant a) the recombination occurred between F4 and 1.4 region either 5'of sat or 3 of sat gene deleting one or both markers. In variant c) the recombination occurred exclusively between F4 and 1.4 region 5'of sat gene. Thus, applying the appropriate selection procedure, the type of 4 marker deletion may be chosen.

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