BACKGROUND OF THE INVENTION Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

The increasing sophistication of recombinant DNA techniques is greatly facilitating research and development in the medical and veterinary industries. One important aspect of recombinant DNA technology is the development of means to alter the genotype by modulating expression of genetic material. A myriad of desirable phenotypic traits are potentially obtainable following selective inactivation of gene expression.

Gene inactivation, that is, the inactivation of gene expression, may occur in cis or in trans. For cis inactivation, only the target gene is inactivated and other similar

genes dispersed throughout the genome are not affected. In contrast, inactivation in trans occurs when one or more genes dispersed throughout the genome and sharing homology with a particular target sequence are also inactivated. In the literature, the term"gene silencing"is frequently used. However, this is generally done without an appreciation of whether the gene silencing events are capable of acting in trans or in cis. This is relevant to the commercial exploitation of gene silencing technology since cis inactivation events are of less usefulness than events in trans. For example, there is less likelihood of success in targeting endogenous genes or exogenous genes (e. g. genes from pathogens) using techniques which promote cis inactivation. Furthermore, in instances where gene inactivation is monitored using a marker gene, it is frequently not possible to discriminate between cis and trans inactivation events. There is, therefore, confusion in the literature regarding the precise molecular mechanisms of gene inactivation (Garrick et al., 1998; Pal-Bahdra et a/., 1997; Bahramian and Zarbl, 1999).

The existing literature is extremely confused as to mechanisms of gene inactivation or gene silencing. For example, the term"antisense"is used to describe situations where genetic constructs designed to express antisense RNAs are introduced into a cell, the aim being to decrease expression of that particular RNA. This strategy has been widely used experimentally and in practical applications. The mechanism by which antisense RNAs function is generally believed to involve duplex formation between the endogenous sense RNA and the antisense sequences which inhibits translation. There is, however, no unequivocal evidence that this mechanism occurs at all in higher eukaryotic systems.

The term"gene silencing"is frequently used to describe inactivation of the expression of a transgene in eukaryotic cells. There is much confusion in the literature as to the mechanism by which this occurs, although it is generally believed to result from transcriptional inactivation. It is unclear whether this particular mechanism has any great practical utility since the expression of the gene itself is inactivated, i. e. there is no trans inactivation of other genes.

In plants, the term"co-suppression"is used to describe precisely situations where a transgene is introduced stably into the genome and expressed as a sense RNA.

Surprisingly, expression of such transgene sequences results in inactivation of homologous genes, i. e. a sequence specific trans inactivation of gene expression (Napoli et a/., 1990; van der Krol et a/., 1990). The molecular phenotype of cells in which this occurs is well described in plant systems: a gene is transcribed as a precursor mRNA, but it is not translated. Another term used to describe co- suppression is post-transcriptional gene inactivation. The disappearance of mRNA sequences is thought to occur as a consequence of activation of a sequence specific RNA degradative system (Lindbo et a/., 1993; Waterhouse et a/., 1999).

There is considerable confusion within the animal literature regarding the term"co- suppression" (Bingham, 1997).

Co-suppression, as defined by the specific molecular phenotype of gene transcription without translation, has previously been considered not to occur in mammalian systems. It has been described only in plant systems and a lower eukaryote, Neurospera (Cogoni et a/., 1996; Cogoni and Macino, 1997).

In work leading up to the present invention, the inventors have employed genetic manipulative techniques to induce gene silencing in animals. The genetic manipulative techniques involve the induction of post-transcriptional inactivation events. The inventors have thereby provided a means for co-suppression in animals. The induction of co-suppression in animals permits the manipulation of a range of phenotypes in animals.

SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as"comprises"or"comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

One aspect of the present invention provides a genetically modified non-human animal having one or more cells that comprise a genetic construct having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation, wherein post-transcriptional expression of the target gene in the cell is repressed, delayed or otherwise reduced.

Another aspect of the present invention provides genetically modified non-human animal having one or more cells in which the genome includes a polynucleotide having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation, wherein post-transcriptional expression of the target gene in the cell is repressed, delayed or otherwise reduced.

In a further aspect of the invention there is provided a transgenic non-human animal in which expression of a target gene is repressed, delayed or otherwise reduced in at least one cell, the cell comprising a genetic construct having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation.

In a further aspect of the present invention there is provided a method of producing a genetically modified animal comprising introducing into the animal a genetic construct having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation.

In another aspect of the present invention there is provided a method of producing a transgenic animal comprising introducing into an animal cell a genetic construct

having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation and allowing the cell to develop into the animal.

Another aspect of the present invention provides a genetically modified mammal comprising a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a cell of said mammal wherein an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product. The mammal may be murine.

Another aspect of the invention provides a transgenic mammal in which expression of a target gene is repressed, delayed or otherwise reduced in a cell, tissue or organ of the mammal to which has been introduced one or more dispersed or foreign nucleic acid molecules which include multiple copies of a nucleotide sequence which is substantially identical to or complementary to the target gene or a region thereof operably under the control of a promoter.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation of the plasmid, pEGFP-N1. For further details, refer to Example 2.

Figure 2 is a diagrammatic representation of the plasmid, pCMV. cass. For further details, refer to Example 2.

Figure 3 is a diagrammatic representation of the plasmid, pCMV. BGI2. cass. For further details, refer to Example 3.

Figure 4 is a diagrammatic representation of the plasmid, pCMV. EGFP. For further details, refer to Example 3.

Figure 5 is a diagrammatic representation of the plasmid, pCMV. TYR. BG12. RYT.

For further details, refer to Example 3.

Figure 6 is a diagrammatic representation of the plasmid, pCMV. TYR. For further details, refer to Example 3.

Figure 7 is a diagrammatic representation of the plasmid, pCMV. TYR. TYR. For further details, refer to Example 3.

Figure 8 shows levels of pigmentation in B16 cells and B16 cells transformed with pCMV. TYR. BG12. RYT. Cell lines are, from left to right: B16, B16 2.1. 6, B16 2.1. 11, B16 3.1. 4, B16 3.1. 15, B16 4.12. 2 and B16 4.12. 3. For further details, refer to Example 3.

Figure 9 is a diagrammatic representation of the plasmid, pCMV. GALT. Bd 12. TLAG. For further details, refer to Example 5.

Figure 10 is a Southern blot of Tyr (hp) transgenic founders. DNA was extracted from tail tip samples of mice born from zygotes injected with a tyrosinase hairpin construct (Tyr (hp) ) into the male pronucleus, as described. Samples were digested with BamHl and probed with the CMV promoter. Mouse #75-038 was identified as transgenic (female).

Figure 11 is a Dot blot of A-generation progeny of Tyr (hp) transgenic founder #75- 038. DNA was extracted from tail tip samples of A-generation progeny A036 and A037, as shown. Dot blot samples were probed with the CMV promoter or an endogenous control sequence (Shiraz 3'), as shown. A-generation mice were bred from mating #75-038 with a C57BI/6 male. Mouse &num 75-A037 was identified as transgenic (female).

Figure 12 is a Southern blot of GaIT (hp) transgenic founders. DNA was extracted from tail tip samples of mice born from zygotes injected with a GaIT hairpin construct (GaIT (hp) ) into the male pronucleus, as described. Samples were digested with BamHl and probed with the CMV promoter. Mice &num 74-026, #74-028,

#74-034 were identified as transgenic (male, male, female, respectively); #74-028 was subsequently revealed to contain two unlined integrations of the construct.

Figure 13 is a Southern blot of A-generation progeny of GaiT (hp) transgenic founder #74-026. DNA was extracted from tail tip samples of A-generation progeny A013, A014 and A015, as shown. Samples were digested with BamHl and probed with the CMV promoter. A-generation mice were bred from mating #74-026 with a C57BI/6 female. Mice &num 74-A013 (male) and &num 74-A015 (male) were identified as transgenic (female).

Figure 14 is a Southern blot of A-generation progeny of GaIT (hp) transgenic founder &num 74-028. DNA was extracted from tail tip samples of A-generation progeny A022-A029, A032 and A033, as shown. Samples were digested with BamHl and probed with the CMV promoter. A-generation mice were bred from mating #74-028 with a C57BI/6 female. Mouse &num 74-A025 (male) was identified as transgenic, containing one of the segregated GaIT (hp) insertions of founder &num 74- 028.

Figure 15 is a FACScan analysis of peripheral blood lymphocytes from GaIT (hp) transgenic mice. Transgenic mice and littermate controls were eye-bled into heparinised tubes (all manipulations were done on ice). Red blood cells were lysed and lymphocytes recovered by centrifugation and fixed in 4% paraformaldehyde in PBS. The cells were dual-labelled for Thy-1 and galactosyl residues with anti-Thy-1 MAb-FITC and lectin IB4-biotin, respectively. After washing, the cells were incubated with streptavidin-Cy5, washed and analysed by dual-channel analysis using a FACScan. Samples from A-generation mice (as in previous figures) 026-A015 and 028-A025 were markedly reduced in lectin binding, as shown, reflecting downregulation of GaIT.

Figure 16 is a photograph of a mouse transformed according to Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention includes the use of sense-antisense nucleotide sequences relative to an endogenous nucleotide sequence in a vertebrate animal cell to down-regulate expression of a gene comprising that endogenous nucleotide sequence. The endogenous nucleotide sequence may comprise all or part of a gene and may or may not be endogenous to the cell. A non-endogenous gene includes a gene in the animal cell introduced by, for example, viral infection or recombinant DNA technology. An endogenous gene includes a gene which would be considered to be naturally present in the animal cell. The down-regulation of a target endogenous gene includes the introduction of the sense nucleotide sequence to that particular cell or a parent of that cell.

Accordingly, one aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal wherein upon introduction of said genetic construct to said animal, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.

Reference to"altered capacity"preferably includes a reduction in the level of translation such as from about 10% to about 100% and more preferably from about 20% to about 90% relative to a cell which is not genetically modified. In a particularly preferred embodiment, the gene corresponding to the target endogenous sequence is substantially not translated into a proteinaceous product.

Conveniently, an altered capacity of translation is determined by any change of phenotype wherein the phenotype, in a non-genetically modified cell, is facilitated by the expression of said endogenous gene.

Preferably the vertebrate animal is derived from mammals, avian species, fish or reptiles. Preferably, the vertebrate animal is derived from mammals, more preferably human, primate, livestock animal (e. g. sheep, cow, goat, pig, donkey,

horse), laboratory test animal (e. g. rat, mouse, rabbit, guinea pig, hamster), companion animal (e. g. dog, cat) or captured wild animal. Particularly preferred mammalian cells are from human and murine animals.

The nucleotide sequence in the genome of a vertebrate animal cell is referred to as a"genomic"nucleotide sequence and preferably corresponds to a gene encoding a product conferring a particular phenotype on the animal cell, group of animal cells and/or an animal comprising said cells. As stated above, the endogenous gene may be endogenous to the animal cell or may be derived from a exogenous source such as a virus, intracellular parasite or introduced by recombinant or other physical means. Reference, therefore, to"genome"or "genomic"includes not only chromosomal genetic material but also extrachromosomal genetic material such as derived from non-integrated viruses.

Reference to a"substantially identical"nucleotide sequence is also encompassed by terms including substantial homology and substantial similarity.

Reference herein to a"gene"is to be taken in its broadest context and includes : - (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i. e. introns, 5'-and 3'-untranslated sequences); (ii) mRNA or cDNA corresponding to the coding regions (i. e. exons) optionally comprising 5'-and 3'-untranslated sequences linked thereto; or (iii) an amplified DNA fragment or other recombinant nucleic acid molecule produced in vitro and comprising all or a part of the coding region and/or 5'- or 3'-untranslated sequences linked thereto.

The gene in the animal genome is also referred to as a target gene or target sequence and may be, as stated above, naturally resident in the genome or may be introduced by recombinant techniques or other means, e. g. viral infection. The term"gene"is not to be construed as limiting the target sequence to any particular structure, size or composition. The target sequence or gene is any nucleotide

sequence which is capable of being expressed to form a mRNA and/or a proteinaceous product. The term"expressed"and related terms such as "expression"include one or both steps of transcription and/or translation.

In a preferred embodiment, the nucleotide sequence in the genetic construct further comprises a nucleotide sequence complementary to the target endogenous nucleotide sequence. Accordingly, another aspect of the present invention provides a genetic construct comprising:- (i) a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell ; (ii) a single nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i); (iii) an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii); wherein upon introduction of said construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for transcription.

Preferably, the identical and complementary sequences are separated by an intron sequence. An example of a suitable intron sequence includes but is not limited to all or part of an intron from a gene encoding ß-globin such as human ß-globin intron 2.

The loss of proteinaceous product is conveniently observed by the change (e. g. loss) of a phenotypic property or an alteration in a genotypic property.

The target gene may encode a structural protein or a regulatory protein. A "regulatory protein"includes a transcription factor, heat shock protein or a protein involved in DNA/RNA replication, transcription and/or translation. The target gene may also be resident in a viral genome which has integrated into the animal gene

or is present as an extrachromosomal element. For example, the target gene may be a gene on an HIV genome. In this case, the genetic construct is useful in inactivating translation of the HIV gene in a mammalian cell.

In a particularly preferred embodiment, the post-transcriptional inactivation is preferably by a mechanism involving trans inactivation.

The genetic construct of the present invention generally, but not exclusively, comprises a synthetic gene. A"synthetic gene"comprises a nucleotide sequence which, when expressed inside an animal, down-regulates expression of a homologous gene, endogenous to the animal or an integrated viral gene resident therein. Preferably, the nucleotide sequence is 20 to 30 nucleotides in length.

A synthetic gene of the present invention may be derived from naturally-occurring genes by standard recombinant techniques, the only requirement being that the synthetic gene is substantially identical or otherwise similar at the nucleotide sequence level to at least a part of the target gene, the expression of which is to be modified. By"substantially identical"is meant that the structural gene sequence of the synthetic gene is at least about 80-90% identical to 30 or more contiguous nucleotides of the target gene, more preferably at least about 90-95% identical to 30 or more contiguous nucleotides of the target gene and even more preferably at least about 95-99% identical or absolutely identical to 30 or more contiguous nucleotides of the target gene. Alternatively, the gene is capable of hybridizing to a target gene sequence under low, preferably medium or more preferably high stringency conditions.

Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium

stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out at Tm = 69.3 + 0.41 (G+C) % (Marmur and Doty, 1962). However, the Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows : low stringency is 6 x SSC buffer, 0. 1% w/v SDS at 25-42°C ; a moderate stringency is 2 x SSC buffer, 0. 1% w/v SDS at a temperature in the range 20°C to 65°C ; high stringency is 0.1 x SSC buffer, 0. 1% w/v SDS at a temperature of at least 65°C.

Generally, a synthetic gene of the instant invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions without affecting its ability to modify target gene expression.

Nucleotide insertional derivatives of the synthetic gene of the present invention include 5'and 3'terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be"silent"in that the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.

Accordingly, the present invention extends to homologs, analogs and derivatives of the synthetic genes described herein.

For the present purpose,"homologs"of a gene as hereinbefore defined or of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogs"of a gene as hereinbefore defined or of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non- nucleotide constituents not normally present in said isolated nucleic acid molecule, for example, carbohydrates, radiochemicals including radionucleotides, reporter molecules such as but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives"of a gene as hereinbefore defined or of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof.

Accordingly, the structural gene component of the synthetic gene may comprise a nucleotide sequence which is at least about 80% identical or homologous to at least about 20 to 30 contiguous nucleotides of an endogenous target gene, a foreign target gene or a viral target gene present in an animal cell or a homologue, analogue, derivative thereof or a complementary sequence thereto.

The genetic construct of the present invention generally but not exclusively comprises a nucleotide sequence, such as in the form of a synthetic gene, operably linked to a promoter sequence. Other components of the genetic construct include but are not limited to regulatory regions, transcriptional start or

modifying sites and one or more genes encoding a reporter molecule. Further components able to be included on the genetic construct extend to viral components such as viral DNA polymerase and/or RNA polymerase. Non-viral components include RNA-dependent RNA polymerase. The structural portion of the synthetic gene may or may not contain a translational start site or 5'-and 3'- untranslated regions, and may or may not encode the full length protein produced by the corresponding endogenous mammalian gene.

Another aspect of the present invention provides a genetic construct comprising a nucleotide sequence substantially homologous to a nucleotide sequence in the genome of a mammalian cell, said first-mentioned nucleotide sequence operably linked to a promoter, said genetic construct optionally further comprising one or more regulatory sequences and/or a gene sequence encoding a reporter molecule wherein upon introduction of said genetic construct into an animal cell, the expression of the endogenous nucleotide sequences having homology to the nucleotide sequence on the genetic construct is inhibited, reduced or otherwise down-regulated via a process comprising post-transcriptional modulation.

Reference herein to a"promoter"is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation in eukaryotic cells, with or without a CCAAT box sequence and additional regulatory elements (i. e. upstream activating sequences, enhancers and silencers).

A promoter is usually, but not necessarily, positioned upstream or 5', of the structural gene component of the synthetic gene of the invention, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the structural gene. In the present context, the term"promoter"is also used to describe a synthetic or fusion molecule or derivative which confers, activates or enhances expression of an isolated nucleic acid molecule in a mammalian cell. Another or the same promoter may also be required to function in plant, animal, insect, fungal, yeast or bacterial cells. Preferred promoters may contain additional copies

of one or more specific regulatory elements to further enhance expression of a structural gene, which in turn regulates and/or alters the spatial expression and/or temporal expression of the gene. For example, regulatory elements which confer inducibility on the expression of the structural gene may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule.

Placing a structural gene under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i. e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i. e. the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

The promoter may regulate the expression of the structural gene component constitutively, or differentially with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to stimuli such as physiological stresses, regulatory proteins, hormones, pathogens or metal ions, amongst others.

Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a mammalian cell, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression of the target gene in said cell.

Promoters may be constitutive, inducible or developmentally regulated.

In the present context, the terms"in operable connection with"or"operably under the control"or similar shall be taken to indicate that expression of the structural gene is under the control of the promoter sequence with which it is spatially connected in a cell.

The genetic construct of the present invention may also comprise multiple nucleotide sequences each optionally operably linked to one or more promoters and each directed to a target gene within the animal cell.

A multiple nucleotide sequence may comprise a tandem repeat or concatemer of two or more identical nucleotide sequences or alternatively, a tandem array or concatemer of non-identical nucleotide sequences, the only requirement being that each of the nucleotide sequences contained therein is substantially identical to the target gene sequence or a complementary sequence thereto. In this regard, those skilled in the art will be aware that a cDNA molecule may also be regarded as a multiple structural gene sequence in the context of the present invention, insofar as it comprises a tandem array or concatemer of exon sequences derived from a genomic target gene. Accordingly, cDNA molecules and any tandem array, tandem repeat or concatemer of exon sequences and/or intron sequences and/or 5'-untranslated and/or 3'-untranslated sequences are clearly encompassed by this embodiment of the invention.

Preferably, the multiple nucleotide sequences comprise at least 2-8 individual structural gene sequences, more preferably at least about 2-6 individual structural gene sequences and more preferably at least about 2-4 individual structural gene sequences.

The optimum number of structural gene sequences which may be involved in the synthetic gene of the present invention will vary considerably, depending upon the length of each of said structural gene sequences, their orientation and degree of identity to each other. For example, those skilled in the art will be aware of the inherent instability of palindromic nucleotide sequences in vivo and the difficulties associated with constructing long synthetic genes comprising inverted repeated

nucleotide sequences, because of the tendency for such sequences to form hairpin loops and to recombine in vivo. Notwithstanding such difficulties, the optimum number of structural gene sequences to be included in the synthetic genes of the present invention may be determined empirically by those skilled in the art, without any undue experimentation and by following standard procedures such as the construction of the synthetic gene of the invention using recombinase- deficient cell lines, reducing the number of repeated sequences to a level which eliminates or minimizes recombination events and by keeping the total length of the multiple structural gene sequence to an acceptable limit, preferably no more than 5-10 kb, more preferably no more than 2-5 kb and even more preferably no more than 0.5-2. 0 kb in length.

In one embodiment, the effect of the genetic construct including synthetic gene comprising the sense nucleotide sequence is to reduce translation of transcript to proteinaceous product while not substantially reducing the level of transcription of the target gene. Alternatively or in addition to, the genetic construct including synthetic gene does not result in a substantial reduction in steady state levels of total RNA.

Accordingly, a particularly preferred embodiment of the present invention provides a genetic construct comprising:- (i) a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell ; (ii) a nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i); (iii) an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii); wherein upon introduction of said construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a

proteinaceous product and wherein there is substantially no reduction in the level of transcription of said gene comprising the endogenous target sequence and/or total level of RNA transcribed from said gene comprising said endogenous target sequence of nucleotides is not substantially reduced.

Preferably, the animal cell is a mammalian cell such as but not limited to a human or murine animal cell.

The present invention further extends to a genetically modified non-human animal having one or more cells that comprise a genetic construct having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation, wherein post-transcriptional expression of the target gene in the cell is repressed, delayed or otherwise reduced.

The animal cell according to this embodiment is preferably from a vertebrate animal. The vertebrate animal cell may be from a mammal, avian species, fish or reptile. More preferably, the animal cell is of mammalian origin such as from a human, primate, livestock animal or laboratory test animal. Particularly preferred animal cells are from human and murine species.

One of the copies of the nucleotide sequence in the sense orientation and one of the copies in the antisense orientation may be separated by a stuffer fragment which comprises a sequence of nucleotides or a homologue, analogue or derivative thereof. Preferably, the stuffer fragment is 10-500 nucleotides in length.

More preferably the stuffer fragment is 50-100 nucleotides in length.

The copies of the nucleotide sequence may be 20-1000 nucleotides in length. In one embodiment the copies of the nucleotide sequence are 20-100 nucleotides in length. In another embodiment, the copies of the nucleotide sequence are 100-500 nucleotides in length.

The nucleotide sequence comprising the sense copy of the target endogenous nucleotide sequence may further comprise a nucleotide sequence complementary to said target sequence. Preferably, the identical and complementary sequences are separated by an intron sequence such as, for example, from a gene encoding p-giobin (e. g. human ß-globin intron 2).

Furthermore, in one embodiment, there is substantially no reduction in levels of steady state total RNA as a result of the introduction of a nucleotide sequence comprising the sense copy of the target sequence.

The present invention further extends to transgenic including genetically modified animal cells and cell lines which exhibit a modified phenotype characterized by a post-transcriptionally modulated genetic sequence.

Accordingly, another aspect of the present invention is directed to an animal comprising said cells wherein the cell or its animal host exhibits at least one altered phenotype compared to the cell or an animal prior to genetic manipulation, said genetic manipulation comprising introducing to an animal cell a genetic construct comprising a nucleotide sequence having substantial homology to a target nucleotide sequence within the genome of said animal cell and wherein the expression of said target nucleotide sequence is modulated at the post- transcriptional level. Preferably, the nucleotide sequence on the genetic construct is operably linked to a promoter.

Optionally, the genetic construct may comprise two or more nucleotide sequences, each operably linked to one or more promoters and each having homology to an endogenous mammalian nucleotide sequence.

The present invention extends to a genetically modified animal such as a mammal comprising one or more cells in which an endogenous gene is substantially transcribed but not translated resulting in a modifying phenotype relative to the animal or cells of the animal prior to genetic manipulation.

In this specification the term"genetically modified animal"is to be taken in its broadest context and includes a transgenic animal, including a transgenic animal capable of producing offspring having substantially the same genetic modification.

Another aspect of the present invention provides a genetically modified murine animal comprising a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a cell of said murine animal wherein an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.

Preferred murine animals are mice and are useful inter alia as experimental animal models to test therapeutic protocols and to screen for therapeutic agents.

In a preferred embodiment, the genetically modified murine animal further comprises a sequence complementary to the target endogenous sequence.

Generally, the identical and complementary sequences may be separated by an intron sequence as stated above.

The present invention further contemplates a method of altering the phenotype of a vertebrate animal cell wherein said phenotype is conferred or otherwise facilitated by the expression of an endogenous gene, said method comprising introducing a genetic construct into said cell or a parent of said cell wherein the genetic construct comprises a nucleotide sequence substantially identical to a nucleotide sequence comprising said endogenous gene or part thereof and wherein a transcript exhibits an altered capacity for translation into a proteinaceous product compared to a cell without having had the genetic construct introduced.

In another aspect of the invention there is provided a method of producing a genetically modified animal comprising introducing into the animal a genetic construct having at least two copies of a nucleotide sequence which is substantially identical to at least a region of a target gene in the cell wherein at

least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation.

The genetic construct may be introduced into the animal by any suitable technique known to those skilled in the art. Such techniques include, but are not limited to transfection of cells, liposome-mediated transformation and microinjection. The genetic construct may also be introduced into the animal by use of a viral vector.

The viral vector may be selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes simplex virus and a pox virus.

Reference herein to homology includes substantial homology and in particular substantial nucleotide similarity and more preferably nucleotide identity.

The term"similarity"as used herein includes exact identity between compared sequences at the nucleotide level. Where there is non-identity at the nucleotide level,"similarity"includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides include"reference sequence", "comparison window","sequence similarity","sequence identity", "percentage of sequence similarity","percentage of sequence identity","substantially similar"and"substantial identity". A"reference sequence"is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides, in length. Because two polynucleotides may each comprise (1) a sequence (i. e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a

"comparison window"to identify and compare local regions of sequence similarity.

A"comparison window"refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i. e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i. e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (1998).

The terms"sequence similarity"and"sequence identity"as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis over a window of comparison. Thus, a"percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e. g. A, T, C, G, !) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention,"sequence identity" will be understood to mean the"match percentage"calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software engineering Co. , Ltd. , South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

The present invention is further directed to the use of genetic construct comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell in the generation of an animal cell wherein an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product. Preferably, the vertebrate animal cell is as defined above and is most preferably a human or murine species.

The construct may further comprise a nucleotide sequence complementary to said target endogenous nucleotide sequence and the nucleotide sequences identical and complementary to said target endogenous nucleotide sequences may be separated by an intron sequence as described above. In one embodiment, there is no reduction in the level of transcription of said gene comprising the endogenous target sequence and/or steady state levels of total RNA are not substantially reduced.

Still a further aspect of the present invention contemplates a method of genetic therapy in a vertebrate animal, said method comprising introducing into cells of said animal comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome of said animal cells such that upon introduction of said nucleotide sequence, RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.

Reference herein to"genetic therapy"includes gene therapy. The genetic therapy contemplated by the present invention further includes somatic gene therapy whereby cells are removed, genetically modified and then replaced into an individual. Preferably, the animal is a human.

Additional information on genetic constructs and methods suitable for use in the present invention is provided in International Patent Application PCT/AU01/00297, the entire contents of which is incorporated herein by reference.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Generic techniques 1. Tissue culture manipulations (a) Adherent cell lines Cells were grown as adherent monolayers. Growth medium consisted of either DMEM (Life Technologies) supplemented with 10% v/v FBS or RPMI 1640 Medium (Life Technologies) supplemented with 10% v/v FBS. Cells were grown in incubators at 37°C in an atmosphere containing 5% v/v C02 in a variety of tissue culture vessels, depending on experimental requirements. The vessels used were: 96-well tissue culture plates (vessels containing 96 separate tissue culture wells each about 0.7 cm in diameter; Costar); 48-well tissue culture plates (vessels containing 48 separate tissue culture wells, each about 1.2 cm in diameter; Costar); 6-well tissue culture plates (vessels containing 6 separate wells, each about 3.8 cm diameter; Nunc); or larger T25 and T75 culture flasks (Nunc).

Medium was changed at 48-72 hr intervals. This was accomplished by removing spent medium, washing the cell monolayers in the tissue culture vessel by adding Phosphate Buffered Saline (1 x PBS; Sigma) and gently rocking the culture vessel, removing the 1 x PBS and adding fresh medium. The volumes of 1 x PBS used in these manipulations were typically 100 pI, 400 pi, 1 ml, 2 ml and 5 ml for 96-well, 48-well, 6-well, T25 and T75 vessels, respectively. Tissue culture media volumes were typically 200 pi for 96-well tissue culture plates, 0.4 ml for 48-well tissue culture plates, 4 mi for 6-well tissue culture plates, 11 ml for T 25 and 40 ml for T75 tissue culture vessels.

Where it was necessary to change culture vessels, monolayers were washed twice with 1 x PBS and then treated with trypsin-EDTA (Life Technologies) for 5 min at 37°C. Under these conditions cells lose adherence and can be

resuspended by trituration and transferred to DMEM, 10% v/v FBS, which stops the action of trypsin-EDTA. The volumes of 1 x PBS for washing and Trypsin- EDTA used for such manipulations were typically 100 gul, 400 iul, 1 ml, 2 ml and 5 ml for 96-well, 48-well, 6-well, T25 and T75 vessels, respectively.

During the course of these experiments it was frequently necessary to passage the cell monolayer. To achieve this, the monolayers were washed twice with 1 x PBS and then treated with trypsin-EDTA for 5 min at 37°C. The volumes of trypsin- EDTA used for such manipulations were typically 20 pI, 100 u !, 500 u), 1 ml and 2 ml for 96 well, 48 well, 6 well, T25 and T75 vessels, respectively. The action of the trypsin-EDTA was stopped with an equal volume of growth medium. The cells were suspended by trituration. A 1/5 volume of the cell suspension was then transferred to a new vessel containing growth medium. Tissue culture medium volumes were typically 192 pi for 96-well tissue culture plates, 360 pi for 48-well tissue culture plates, 3.8 ml for 6-well tissue culture plates, 9.6 ml for T25 and 39.2 ml for T75 tissue culture vessels.

Cell suspensions were counted microscopically using a haemocytometer (Hawksley).

(b) Non-adherent cells Non-adherent cells were grown in growth medium similarly to adherent cell lines.

As in the case of adherent monolayers, frequent changes of tissue culture vessels were necessary. For T25 and T75 vessels, the cell suspension was removed to 50 ml sterile plastic tubes (Falcon) and centrifuged for 5 min at 500 x g and 4°C. The supernatant was then discarded and the cell pellet suspended in growth medium.

The cell suspension was then placed into a new tissue culture vessel. For 96-well, 48-well, and 6-well vessels, the vessels were centrifuged for 5 min at 500 x g and 4°C. The supernatant was then aspirated away from the cell pellet and the cells suspended in growth medium. The cells were then transferred to a new tissue culture vessel. Tissue culture media volumes were typically 200 pi for 96-well

tissue culture plates, 400, ul for 48-well tissue culture plates, 4 ml for 6-well tissue culture plates, 11 ml for T25 and 40 mi for T75 tissue culture vessels.

Passaging the cell suspensions was achieved in the following manner. Cells were centrifuged for 5 min at 500 x g and 4°C and suspended in 5 ml growth medium.

Then 0.5 ml (T25) or 1.0 ml (T75) of the cell suspension was transferred to a new vessel containing growth medium. For cells in 96-well, 48-well, and 6-well plates, a 1/5 volume of cells was transferred to the corresponding wells of a new vessel containing 4/5 volume of growth medium. Cells were counted as described for adherent cells.

2. Protocol for freezing cells Cells stored for later use were frozen. Adherent monolayers were washed twice with 1 x PBS and then treated with trypsin-EDTA (Life Technologies) for 5 min at 37°C. Non-adherent cells were centrifuged for 5 min at 500 x g and 4°C. The cells were suspended by trituration and transferred to storage medium consisting of DMEM RPMI 1640 supplemented with 20% v/v FBS and 10% v/v dimethylsulfoxide (Sigma).

Aliquots of cells were transferred to 1.5 mi cryotubes (Nunc) and the tubes were placed in a Cryo 1° freezing container (Nalgene) containing propan-2-01 (BDH) and cooled slowly to-70°C. The tubes of cells were stored at-70°C. Reanimation of stored cells was achieved by warming the cells to O°C on ice. The cells were then transferred to a T25 flask containing DMEM and 20% v/v FBS, and then incubated at 37°C in an atmosphere of 5% v/v C02, 3. Cloning of cell lines Adherent and non-adherent mammalian cell types were transfected with specific plasmid vectors carrying expression constructs to target specific genes of interest.

Stable, transformed cell colonies were selected over a period of 2-3 weeks using cell growth medium (either DMEM, 10% v/v FBS or RPMI 1640,10% v/v FBS)

supplemented with geneticin or puromycin. Individual colonies were cloned to establish new transfected cell lines.

(a) Adherent cells In examples using adherent cells, individual lines were cloned from discrete colonies as follows. First, the medium was removed from an individual well of a 6- well tissue culture vessel and the cell colonies washed twice with 2 mi of 1 x PBS.

Next, individual colonies were detached from the plastic culture vessel with a sterile plastic pipette tip and moved to a 96-well plate containing 200 ul of conditioned medium supplemented with either geneticin or puromycin. The vessel was then incubated at 37°C and 5% v/v C02 for approximately 72 hr. Individual wells were microscopically examined for growing colonies and the medium replaced with fresh growth medium. When the monolayer of each stable line had reached about 90% confluency it was transferred in successive steps as previously described until the stable, transformed line was housed in a T25 tissue culture vessel. At this point, aliquots of each stable cell line were frozen for long term maintenance.

(b) Non-adherent cells Non-adherent cells were cloned by dilution cloning. Transformed cells were biologically cloned using a dilution strategy, whereby colonies were established from single cells. To support growth of single colonies,"conditioned media"were used. Conditioned media were prepared by overlaying 20-30% confluent monolayers of PK-1 cells grown in a T75 vessel with 40 ml of DMEM containing 10% v/v FBS. Vessels were incubated at 37°C, 5% v/v C02 for 24 hr, after which the growth medium was transferred to a sterile 50 ml tube (Falcon) and centrifuged at 500 x g. The growth medium was passed through a 0. 45 um filter and decanted to a fresh sterile tube and used as"conditioned medium".

A T75 vessel containing mixed colonies of transformed PK-1 cells at 20-30% confluency was washed twice with 1 x PBS and cells separated by trypsin

treatment as described above, then diluted into 10 mi of DMEM, 10% v/v FBS. The cell concentration was determined microscopically using a haemocytometer slide and cells diluted to 10 cells per ml in conditioned medium. Single wells of 96-well tissue culture vessels were seeded with 200 pi of the diluted cells in conditioned medium and cells were incubated at 37°C and 5% v/v C02 for 48 hr. Wells were inspected microscopically and those containing a single colony, arising from a single cell, were defined as clonal cell lines. The original conditioned medium was removed and replaced with 200 ul of fresh conditioned medium and cells incubated at 37°C and 5% v/v C02 for 48 hr. After this time, conditioned medium was replaced with 200 Zul of DMEM, 10% v/v FBS and 1.5 mg/l genetecin and cells again incubated at 37°C and 5% v/v C02. Colonies were allowed to expand and medium was changed every 48 hr.

When the monolayer in an individual well was about 90% confluent, the cells were washed twice with 100 ul of 1 x PBS and cells loosened by treatment with 20 pi of 1 x PBS/1 x trypsin-EDTA as described above. Cells in a single well were transferred to a single well of a 48-well culture vessel containing 500 ul of DMEM, 10% v/v FBS and 1. 5 ug/ml genetecin. Medium was changed every 48-72 hr as hereinbefore described.

When a monolayer in an individual well of a 48-well culture vessel was about 90% confluent, the cells were transferred to 6-well tissue culture vessels using trypsin- EDTA treatment as described above. Separated cells were then transferred to 4 mi DMEM, 10% v/v FBS, 1. 5 ug/ml geneticin and transferred to a single well of a 6-well tissue culture vessel. Cells were grown at 37°C and 5% v/v CO2 and colonies were allowed to expand. Medium was changed every 48 hr.

When monolayers in 6-well culture vessels were about 90% confluent, cells were transferred to T25 vessels using trypsin-EDTA as described above. When these monolayers were about 90% confluent, cells were transferred to T75 culture vessels, as described above. Once individual lines were established in T75 vessels they were either maintained by passaging every 48-72 hr using a one- tenth dilution, or maintained as frozen stocks.

4. Cell nuclei isolation protocol (a) Adherent cells A 100 mm Petri dish (Costar) or T75 flask containing 30 ml of growth medium (DMEM or RPMI 1640) including 10% v/v FBS was seeded with 4 x 106 cells and incubated at 37°C and 5% v/v C02 until the monolayer was about 90% confluent (overnight). The Petri dish containing the monolayer was placed on a bed of ice and chilled before processing. Medium was decanted and 8 ml of 1 x PBS (ice cold) was added to the Petri dish, and the tissue monolayer washed by gently rocking the dish. The PBS was again decanted and the wash repeated.

The tissue monolayer was overlaid with 4 mi of ice-cold sucrose buffer A [0.32 M sucrose; 0.1 mM EDTA; 0. 1% v/v Igepal ; 1.0 mM DTT; 10 mM Tris-HCI, pH 8.0 ; 0.1 mM PMSF; 1.0 mM EGTA; 1.0 mM Spermidine] and cells lysed by incubating them on ice for 2 min. Using a cell scraper, adherent cells were dislodged and a small aliquot of cells examined by phase-contrast microscopy. If the cells had not lysed, they were transferred to an ice-cold dounce homogenizer (Braun) and broken with 5-10 strokes of a type S pestle. Additional strokes were sometimes required. Cells were then examined microscopically to verify that nuclei were free from cytoplasmic debris. Ice-cold sucrose buffer B [1. 7 M sucrose; 5.0 mM magnesium acetate; 0.1 mM EDTA; 1.0 mM DTT. 10 mM Tris-HCI, pH 8.0 ; 0.1 mM PMSF] (4 mi) was then added to the Petri dish and the buffers mixed by gentle stirring with the cell scraper. The final concentration of sucrose in cell homogenates should be sufficient to prevent a large build-up of debris at the interface between the homogenate and the sucrose cushion. The amount of sucrose buffer 2 added to cell homogenate may need to be adjusted accordingly.

(b) Non-adherent cells A T75 tissue culture vessel containing 30 mi of growth medium (DMEM or RPMI 1640) including 10% v/v FBS was seeded with 4 x 106 cells and incubated at 37°C and 5% v/v C02 overnight.

The contents of the T75 flask were transferred to a 50 ml screw-capped tube (Falcon), which was placed on ice and allowed to chill before processing. The tube was centrifuged at 500 x g for 5 min in a chilled centrifuge to pellet cells. Medium was decanted, 10 ml of 1 x PBS (ice cold) added to the tube and the cells suspended by gentle trituration. The PBS was again decanted and the wash repeated.

Cells were suspended in 4 ml of ice-cold sucrose buffer A and lysed by incubating on ice for 2 min and, optionally, by dounce homogenisation, as described above for adherent cells lines.

(c) Isolation protocol To analyze the status of transcription of individual genes in cloned transformed cell lines, nuclear run-on assays were performed. A monolayer of cells was established by seeding a T75 culture vessel with 4 x 106 transformed cells into 40 ml of DMEM, 10% v/v FBS and incubating cells until the monolayer was about 90% confluent. The monolayers were washed twice with 5 mi of 1 x PBS, separated by treatment with 2 ml trypsin-EDTA and transferred to 2 mi of DMEM including 10% v/v FBS.

These cells were transferred to a 10 ml capped tube, 3 mi of ice-cold 1 x PBS was added and the contents mixed by inversion. Transformed PK-1 cells were collected by centrifugation at 500 x g for 10 min at 4°C, the supernatant was discarded and cells were resuspended in 3 ml of ice-cold 1 x PBS by gentle vortexing. Total cell numbers were determined using a haemocytometer; a maximum of 2 x 108 cells was used for subsequent analyses.

Transformed PK-1 cells were collected by centrifugation at 500 x g for 10 min at 4°C and resuspended in 4 ml Sucrose buffer A. Cells were incubated at 4°C for 5 min to allow them to lyse then small aliquots were examined by phase-contrast microscopy. Under these conditions lysis can be visualized. Homogenates were transferred to 50 mi tubes containing 4 ml of ice-cold Sucrose buffer B.

To obtain efficient transcription run-on assays, nuclei should be purified from other cellular debris. One method for this is to purify nuclei by ultra-centrifugation through sucrose pads. The final concentration of sucrose in a cell homogenate should be sufficient to prevent a large build up of debris at the interface between homogenate and the sucrose cushion. Therefore, the amount of Sucrose buffer B added to the initial cell homogenate was varied in some instances.

To prepare a sucrose pad, 4.4 ml ice-cold Sucrose buffer B was transferred to a polyallomer SW41 tube (Beckman). Nuclear preparations were carefully layered over the sucrose pad and centrifuged for 45 min at 30,000 x g (13,300 rpm in SW41 rotor) at 4°C. The supernatant was removed and the pelleted nuclei loosened by gentle vortexing for 5 seconds. Nuclei were resuspended by trituration in 200 pi ice cold glycerol storage buffer (50 mM Tris-HCI (pH 8.3), 40% v/v glycerol, 5 mM magnesium chloride, 0.1 mM EDTA) per 5 x 107 nuclei. One hundred microlitres of this suspension (approximately 2.5 x 107 nuclei) was aliquote into chilled microcentrifuge tubes and 1 pl (40 U) RNasin (Promega) was added. Usually such extracts were used immediately for transcription run-on assays, although they could be frozen on dry ice and stored at-70°C or in liquid nit.

5. Nuclear transcription run-on protocol [a-32P]-UTP-labelled nascent RNA transcripts for gene-specific detection by filter hybridization were prepared as follows : All NTPs were obtained from Roche. Nuclear run-on reactions were initiated by adding 100 pi of 1 mM ATP, 1 mM CTP, 1 mM GTP, 5 mM DTT and 5 pi (50 uCi) [(x32P]-UTP (GeneWorks) to 100 ul of isolated nuclei, prepared as hereinbefore described. The reaction mix was incubated at 30°C for 30 min with shaking and terminated by adding 400 ut of 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 100 mM 2-mercaptoethanol and 0. 5% v/v N-lauryl sarcosine (Solution D).

To purify in vitro synthesized RNAs, 60 ul 2 M sodium acetate (pH 4.0) and 600 ul water-saturated phenol was added and the mixture vortexed; an additional 120 pi

chloroform/isoamylalcohol (49: 1) was added, the mixture vortexed and phases separated by centrifugation.

The aqueous phase was decanted to a fresh tube and 20 ug tRNA added as a carrier. RNA was precipitated by the addition of 650 ul isopropanol and incubation at-20°C for 10 min. RNA was collected by centrifugation at 12,000 rpm at 4°C for 20 min and the pellet was rinsed with cold 70% v/v ethanol. The pellet was dissolved in 30 ut of TE pH 7.3 (10 mM Tris-HCI, 1 mM EDTA) and vortexed to resuspend the pellet. 400 pl of Solution D was added and the mixture vortexed.

The RNA was precipitated by the addition of 430 ul of isopropanol, incubation at- 20°C for 10 mins and centrifuged at 10,000 g for 20 mins at 4°C. The supernatant was removed and the RNA pellet washed with 70% v/v ethanol. The pellet was resuspended in 200 ul of 10 mM Tris (pH 7.3), 1 mM EDTA and incorporation estimated with a hand-held geiger counter.

To prepare the radioactive RNAs for hybridization, samples were precipitated by adding 20 pi 3 M sodium acetate pH 5.2, 500 ul ethanol and collected by centrifugation at 12,000 x g and 4°C for 20 min. The supernatant was removed and the pellet resuspended in 1.5 ml of hybridization buffer (MRC #HS 114F, Molecular Research Centre Inc.).

To detect gene-specific transcription run-on products, an alternative approach to filter hybridization is the ribonuclease protection assay. Strand-specific, gene- specific unlabelled RNA probes are prepared using standard techniques. These are annealed to 32P-labelled RNAs isolated from transcription run-on experiments.

To detect double-stranded RNA, annealing reaction products are treated with a mixture of single strand specific RNases and reaction products are examined using PAGE. Techniques for this are well known to those experienced in the art and are described in RPA III (trademark) handbook'Ribonuclease Protection Assay' (Catalog #s 1414,1415 ; Ambion Inc.).

An additional method was used for the preparation of biotin-labelled nascent RNA transcripts (Patrone et al., 2000) for gene specific detection by real-time PCR

assays. Intact nuclei were isolated from adherent and non-adherent cell types (refer to Examples 12-19, below) and stored at-70°C in concentrations of 1 x 108 per ml in glycerol storage buffer [50 mM Tris-HCI, pH 8.3 ; 40% v/v glycerol, 5 mM MgCI2 and 0. 1 mM EDTA].

One hundred microlitres of nuclei (107) in glycerol storage buffer was added to 100 pl of ice cold reaction buffer supplemented with nucleotides [200 mM KCI, 20 mM Tris-HCI pH 8. 0,5 mM MgCI2, 4 mM dithiothreitol (DTT), 4 mM each of ATP, GTP and CTP, 200 mM sucrose and 20% v/v glycerol]. Biotin-16-UTP (from 10 mM tetralithium salt ; Sigma) was supplied to the mixture, which was incubated for 30 min at 29°C. The reaction was stopped, the nuclei Iysed and digestion of DNA initiated by the addition of 20 ut of 20 mM calcium chloride (Sigma) and 10 pi of 10 mg/ml RNase-free DNase I (Roche). The mixture was incubated for 10 min at 29°C.

Isolation of nuclear run-on and total, including cytoplasmic, RNA was performed using TRlzol (registered trademark) reagent (Life Technologies) as per the manufacturer's instructions. RNA was suspended in 50 ul of RNase-free water.

Nascent biotin-16-UTP-labelled run-on transcripts are then purified from total RNA using streptavidin beads (Dynabeads (registered trademark) kilobaseBINDER (trademark) Kit, Dynal) according to the manufacturer's instructions.

Real-time PCR reactions are performed to quantify gene transcription rates from these run-on experiments. Real-time PCR chemistries are known to those familiar with the art. Sets of oligonucleotide primers are designed which are specific for transgenes, endogenous genes and ubiquitously-expressed control sequences.

Oligonucleotide amplification and reporter primers are designed using Primer Express software (Perkin Elmer). Relative transcript levels are quantified using a Rotor-Gene RG-2000 system (Corbett Research).

6. Detection of mRNA Ribonuclease protection assay, using the method of annealing unlabelled mRNA to 32P-labelled probes, may be used to detect transcripts of endogenous genes and transgenes in the cytoplasm. Reaction products are examined using PAGE.

Steady state levels of RNA products of endogenous genes and transgenes are assessed by Northern analysis.

Alternatively, relative mRNA levels are quantified using real-time PCR with a Rotor-Gene RG-2000 system with amplification and reporter oligonucleotides designed using Primer Express software for specific transgenes, endogenous genes and ubiquitously-expressed control genes.

7. Southern blot analysis of mammalian genomic DNA For all subsequent examples, Southern blot analyses of genomic DNA were carried out according to the following protocol. A T75 tissue culture vessel containing 40 mi of DMEM or RPMI 1640, 10% v/v FBS was seeded with 4 x 106 cells and incubated at 37°C and 5% v/v C02 for 24 hr.

(a) Adherent cells For adherent cells, proceed as follows : decant medium and add 5 ml of 1 x PBS to the T75 flask and wash the tissue monolayer by gently rocking. Decant the PBS and repeat washing of the tissue monolayer with 1 x PBS. Decant the PBS.

Overlay the monolayer with 2 mi 1 x PBS/1 x Trypsin-EDTA. Cover the surface of the tissue monolayer evenly by gentle rocking of the flask. Incubate the T75 flask at 37°C and 5% v/v C02 until the tissue monolayer separates from the flask. Add 2 ml of medium including 10% v/v FBS to the flask. Under microscopic examination, the cells should now be single and round. Transfer the cells to a 10 ml capped tube and add 3 ml of ice-cold 1 x PBS. Invert the tube several times to mix. Pellet the cells by centrifugation at 500 x g for 10 min in a refrigerated centrifuge (4°C).

Decant the supernatant and add 5 mi of ice-cold 1 x PBS to the capped tube.

Suspend the cells by gentle vortexing. Determine the total number of cells using a haemocytometer slide. Cell numbers should not exceed 2 x 108. Pellet the cells by centrifugation at 500 x g for 10 min in a refrigerated centrifuge (4°C). Decant the supernatant.

(b) Non-adherent cells For non-adherent cells proceed as follows : decant cell suspension into a 50 mi Falcon tube and centrifuge at 500 x g for 10 min in a refrigerated centrifuge (4°C).

Decant the supernatant and add 5 mi of ice-cold 1 x PBS to the cells and suspend the cells by gentle vortexing. Pellet the cells by centrifugation at 500 x g for 10 min in a refrigerated centrifuge (4°C). Decant the supernatant and add 5 ml of ice-cold 1 x PBS to the Falcon tube. Suspend the cells by gentle vortexing. Determine the total number of cells using a haemocytometer slide. Cell numbers should not exceed 2 x 108. Pellet the cells by centrifugation at 500 x g for 10 min in a refrigerated centrifuge (4°C). Decant the supernatant.

(c) DNA extraction and analysis Genomic DNA, for both adherent and non-adherent cell lines, was extracted using the Qiagen Genomic DNA extraction kit (Cat No. 10243) as per the manufacturer's instructions. The concentration of genomic DNA recovered was determined using a Beckman model DU64 photospectrometer at a wavelength of 260 nm.

Genomic DNA (10 pg) was digested with appropriate restriction endonucleases and buffer in a volume of 200 ul at 37°C for approximately 16 hr. Following digestion, 20 ut of 3 M sodium acetate pH 5.2 and 500 pi of absolute ethanol were added to the digest and the solutions mixed by vortexing. The mixture was incubated at-20°C for 2 hr to precipitate the digested genomic DNA. The DNA was pelleted by centrifugation at 10,000 x g for 30 min at 4°C. The supernatant was removed and the DNA pellet washed with 500 ut of 70% v/v ethanol. The 70% v/v ethanol was removed, the pellet air-dried, and the DNA suspended in 20 pl of water.

Gel loading dye (0.25% w/v bromophenol blue (Sigma); 0.25% w/v xylene cyanol FF (Sigma); 15% w/v Ficoll Type 400 (Pharmacia)) (5 pi) was added to the resuspended DNA and the mixture transferred to a well of 0.7% w/v agarose/TAE gel containing 0. 5 ug/ml of ethidium bromide. The digested genomic DNA was electrophoresed through the gel at 14 volts for approximately 16 hr. An appropriate DNA size marker was included in a parallel lane.

The digested genomic DNA was then denatured (1.5 M NaCI, 0.5 M NaOH) in the gel and the gel neutralized (1.5 M NaCI, 0.5 M Tris-HCI pH 7.0). The electrophoresed DNA fragments were then capillary blotted to Hybond NX (Amersham) membrane and fixed by UV cross-linking (Bio Rad GS Gene Linker).

The membrane containing the cross-linked digested genomic DNA was rinsed in sterile water. The membrane was then stained in 0.4% v/v methylene blue in 300 mM sodium acetate (pH 5.2) for 5 min to visualize the transferred genomic DNA.

The membrane was then rinsed twice in sterile water and destained in 40% v/v ethanol. The membrane was then rinsed in sterile water to remove ethanol.

The membrane was placed in a Hybaid bottle and 5 ml of pre-hybridization solution added (6 x SSPE, 5 x Denhardt's reagent, 0.5% w/v SDS, 100 iug/ml denatured, fragmented herring sperm DNA). The membrane was pre-hybridized at 60°C for approximately 14 hr in a hybridization oven with constant rotation (6 rpm).

Probe (25 ng) was labelled with [a32P]-dCTP (specific activity 3000 Ci/mmol) using the Megaprime DNA labelling system as per the manufacturer's instructions (Amersham Cat. No. RPN1606). Labelled probe was passed through a G50 Sephadex Quick Spin (trademark) column (Roche, Cat. No. 1273973) to remove unincorporated nucleotides as per the manufacturer's instructions.

The heat-denatured labelled probe was added to 2 ml of hybridization buffer (6 x SSPE, 0.5% w/v SDS, 100 ug/ml denatured, fragmented herring sperm DNA) pre- warmed to 60°C. The pre-hybridization buffer was decanted and replaced with 2 mi of pre-warmed hybridization buffer containing the labelled probe. The

membrane was hybridized at 60°C for approximately 16 hr in a hybridization oven with constant rotation (6 rpm).

The hybridization buffer containing the probe was decanted and the membrane subjected to several washes: 2 x SSC, 0.5% w/v SDS for 5 min at room temperature; 2 x SSC, 0. 1 % w/v SDS for 15 min at room temperature; 0.1 x SSC, 0.5% w/v SDS for 30 min at 37°C with gentle agitation; 0.1 x SSC, 0.5% w/v SDS for 1 hour at 68°C with gentle agitation; and 0.1 x SSC for 5 min at room temperature with gentle agitation.

Washing duration at 68°C varied based on the amount of radioactivity detected with a hand-held Geiger counter.

The damp membrane was wrapped in plastic wrap and exposed to X-ray film (Curix Blue HC-S Plus, AGFA) for 24 to 48 hr and the film developed to visualize bands of probe hybridized to genomic DNA.

EXAMPLE 2 Preparation of plasmid construct cassettes for use in achieving co- suppression 1. Generic RNA isolation, cDNA synthesis and PCR protocol Total RNA was purified from the indicated cell lines using an RNeasy Mini Kit according to the manufacturer's protocol (Qiagen). To prepare cDNA, this RNA was reverse transcribed using Omniscript Reverse Transcriptase (Qiagen). Two

micrograms of total RNA was reverse transcribed using 1 uM oligo dT (Sigma) as a primer in a 20 pi reaction according to the manufacturer's protocol (Qiagen).

To amplify specific products, 2 pi of this mixture was used as a substrate for PCR amplification, which was performed using HotStarTaq DNA polymerase according to the manufacturer's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95°C for 15 mins, followed by 35 amplification cycles of 94°C for 30 secs, 60°C for 30 secs and 72°C for 60 secs, with a final elongation step at 72°C for 4 mins.

PCR products to be cloned were usually purified using a QiAquick PCR Purification Kit (Qiagen); in instances where multiple fragments were generated by PCR, the fragment of the correct size was purified from agarose gels using a QlAquick Gel Purification Kit (Qiagen) according to the manufacturer's protocol.

Amplification products were then cloned into pCR (registered trademark) 2.1-TOPO (Invitrogen) according to the manufacturer's protocol.

2. Generic cloning techniques To prepare the constructs described below, insert fragments were excised from intermediate vectors using restriction enzymes according to the manufacturer's protocols (Roche) and fragments purified from agarose gels using QlAquick Gel Purification Kits (Qiagen) according to the manufacturer's protocol. Vectors were usually prepared by restriction digestion and treated with Shrimp Alkaline Phosphatase according to the manufacturer's protocol (Amersham). Vector and inserts were ligated using T4 DNA ligase according to the manufacturer's protocols (Roche) and transformed into competent E. coli strain DH5a using standard procedures (Sambrook et a/. ; 1984).

3. Constructs (a) Commercial plasmids Plasmid pEGFP-N1 Plasmid pEGFP-N1 (Figure 1; Clontech) contains the CMV IE promoter operably connected to an open reading frame encoding a red-shifted variant of the wild-type GFP which has been optimized for brighter fluorescence. The specific GFP variant encoded by pEGFP-N1 has been disclosed by Cormack et a/. (1996). Plasmid pEGFP-N1 contains a multiple cloning site comprising Bglll and BamHl sites and many other restriction endonuclease cleavage sites, located between the CMV IE promoter and the EGFP open reading frame. The plasmid pEGFP-N1 will express the EGFP protein in mammalian cells. In addition, structural genes cloned into the multiple cloning site will be expressed as EGFP fusion polypeptides if they are in- frame with the EGFP-encoding sequence and lack a functional translation stop codon. The plasmid further comprises an SV40 polyadenylation signal downstream of the EGFP open reading frame to direct proper processing of the 3'- end of mRNA transcribed from the CMV IE promoter sequence (SV40 pA). The plasmid further comprises the SV40 origin of replication functional in animal cells ; the neomycin-resistance gene comprising SV40 early promoter (SV40-E in Figure 1) operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 (Kan/Neo in Figure 1) and the HSV thymidine kinase polyadenylation signal, for selection of transformed cells on kanamycin, neomycin or geneticin; the pUC19 origin of replication which is functional in bacterial cells and the f1 origin of replication for single-stranded DNA production.

Plasmid pCR (registered trademark) 2. 1-TOPO Plasmid pCR (registered trademark) 2.1-TOPO is a commercially available T- tailed vector from Invitrogen and comprises the lack promoter sequence and lacZ- a transcription terminator, with multiple restriction endonuclease cloning sites located there between. Plasmid pCR (registered trademark) 2.1-TOPO is provided

with covalently bound topoisomerase I enzyme for fast cloning. The plasmid further comprises the Co (Ef and fi origins of replication and the kanamycin and ampicillin resistance genes.

(b) Plasmid cassettes Plasmid pCMV. cass Plasmid pCMV. cass (Figure 2) is an expression cassette for driving expression of a structural gene sequence under control of the CMV-IE promoter sequence.

Plasmid pCMV. cass was derived from pEGFP-N1 (Figure 1) by deletion of the EGFP open reading frame as follows : Plasmid pEGFP-N1 was digested with PinAl and Notl, blunt-ended using Pful DNA polymerase and then religated. Structural gene sequences are cloned into pCMV. cass using the multiple cloning site, which is identical to the multiple cloning site of pEGFP-N1, except it lacks the PinAI site.

Plasmid pCMV. BGI2. cass To create pCMV. BGI2. cass (Figure 3), the human ß-globin intron sequence was isolated as a SacUPstl fragment from TOPO. BG12 and cloned between the Sacl and Pstl sites of pCMV. cass. In pCMV. BG) 2. cass, any RNAs transcribed from the CMV promoter will include the human p-giobin intron 2 sequences; these intron sequences will presumably be excised from transcripts as part of the normal intron processing machinery, since the intron sequences include both the splice donor and splice acceptor sequences necessary for normal intron processing.

EXAMPLE 3 Co-suppression of Tyrosinase in Murine Type B16 cells in vitro 1. Culturing of cell lines B16 cells derived from murine melanoma (ATCC CRL-6322) were grown as adherent monolayers using RPMI 1640 supplemented with 10% v/v FBS, as described in Example 1, above.

2. Preparation of genetic constructs (a) Interim plasmid Plasmid TOPO. TYR Total RNA was purified from cultured murine B16 melanoma cells and cDNA prepared as described in Example 2.

To amplify a region of the murine tyrosinase gene, 2 pi of this mixture was used as a substrate for PCR amplification using the primers: TYR-F: GTT TCC AGA TCT CTG ATG GC and TYR-R: AGT CCA CTC TGG ATC CTA GG.

The PCR amplification was performed using HotStarTaq DNA polymerase according to the manufacturer's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95°C for 15 mins, followed by 35 amplification cycles of 94°C for 30 secs, 55°C for 30 secs and 72°C for 60 secs, with a final elongation step at 72°C for 4 mins.

The PCR amplified region of tyrosinase was column purified (PCR purification column, Qiagen) and then cloned into pCR (registered trademark) 2.1-TOPO according to the manufacturer's instructions (Invitrogen) to make plasmid TOPO. TYR.

(b) Test plasmids Plasmid pCMV. EGFP Plasmid pCMV. EGFP (Figure 4) is capable of expressing the entire EGFP open reading frame and is used in this and subsequent examples as a positive transfection control.

Plasmid pCMV. TYR. BG12. RYT Plasmid pCMV. TYR. BG12. RYT (Figure 5) contains an inverted repeat, or palindrome, of a region of the murine tyrosinase gene that is interrupted by the insertion of the human ß-globin intron 2 sequence therein. Plasmid pCMV. TYR. BG12. RYT was constructed in successive steps: (i) the TYR sequence from plasmid TOPO. TYR was sub-cloned in the sense orientation as a Bg/ll-to- BamHl fragment into Bg/ll-digested pCMV. BG) 2 to make plasmid pCMV. TYR. BGI2, and (ii) the TYR sequence from plasmid TOPO. TYR was sub- cloned in the antisense orientation as a Bglll-to-BamHl fragment into BamHl- digested pCMV. TYR. BGI2 to make plasmid pCMV. TYR. BG12. RYT.

Plasmid pCMV. TYR Plasmid pCMV. TYR (Figure 6) contains a single copy of mouse tyrosinase cDNA sequence, expression of which is driven by the CMV promoter. Plasmid pCMV. TYR was constructed by cloning the TYR sequence from plasmid TOPO. TYR as a BamHl-to-Bglll fragment into BamHl-digested pCMV. cass and selecting plasmids containing the TYR sequence in a sense orientation relative to the CMV promoter.

Plasmid pCMV. TYR. TYR Plasmid pCMV. TYR. TYR (Figure 7) contains a direct repeat of the mouse tyrosinase cDNA sequence, expression of which is driven by the CMV promoter.

Plasmid pCMV. TYR. TYR was constructed by cloning the TYR sequence from plasmid TOPO. TYR as a BamHl-to-Bglll fragment into BamHl-digested pCMV. TYR and selecting plasmids containing the second TYR sequence in a sense orientation relative to the CMV promoter.

3. Detection of co-suppression phenotype (a) Reduction of melanin pigmentation through PTGS of tyrosinase by insertion of a region of the tyrosinase gene into murine melanoma B16 cells Tyrosinase is the major enzyme controlling pigmentation in mammals. If the gene is inactivated, melanin will no longer be produced by the pigmented B16 melanoma cells. This is essentially the same process that occurs in albino animals.

Transformations were performed in 6 well tissue culture vessels. Individual wells were seeded with 1 x 105 cells in 2 mi of RPMI 1640, 10% v/v FBS and incubated at 37°C, 5% v/v C02 until the monolayer was 60-90% confluent, typically 16 to 24 hr.

Transformations were performed in 6-well tissue culture vessels. Individual wells were seeded with 2 x 105 B16 cells in 2 mi of DMEM, 10% v/v DCS and incubated at 37°C, 5% v/v C02 until the monolayer was 60-90% confluent, typically 16 to 24 hr.

The following solutions were prepared in 10 mi sterile tubes: Solution A: For each transfection, 1 pLg of DNA (pCMV. BEV2. BG12. 2VEB or pCMV. EGFP-Transfection Control) was diluted into 100 pi of OPTI-

MEM-I (registered trademark) Reduced Serum Medium (serum-free medium) and; Solution B: For each transfection, 10 ul of LiPOFEcTAMINE (trademark) Reagent was diluted into 100 PI OPTi-MEM-I (registered trademark) Reduced Serum Medium.

The two solutions were combined and mixed gently, and incubated at room temperature for 45 min to allow DNA-liposome complexes to form. While complexes formed, the B16 cells were rinsed once with 2 ml of OPTI-MEM I (registered trademark) Reduced Serum Medium.

For each transfection, 0. 8 ml of OPTE-ME I (registered trademark) Reduced Serum Medium was added to the tube containing the complexes, the tube mixed gently, and the diluted complex solution overlaid onto the rinsed B16 cells. Cells were then incubated with the complexes at 37°C and 5% v/v C02 typically for 3 to 4 hours.

Transfection mixture was then removed and the B16 monolayers overlaid with 2 ml of DMEM, 10% v/v DCS. Cells were incubated at 37°C and 5% v/v C02 for approximately 48 hr. To select for stable transformants, the medium was replaced every 72 hr with 4 ml of DMEM, 10% v/v DCS, 0.6 mg/ml geneticin. Cells transformed with the transfection control pCMV. EGFP were examined after 24-48 hr for transient EGFP expression using fluorescence microscopy at a wavelength of 500-550 nm. After 21 days of selection, stably transformed CRIB-1 colonies were apparent.

Individual colonies of stably transfected B16 cells were cloned, maintained and stored as described in Generic Techniques in Example 1, above.

Thirty six clones stably transformed with pCMV. TYR. BG12. RYT, 34 clones stably transformed with pCMV. TYR and 37 clones stably transformed with pCMV. TYR. TYR were selected for subsequent analyses.

When the endogenous tyrosinase gene is post-transcriptionally silenced, melanin production in the B16 cells is reduced. B16 cells that would normally appear to contain a dark brown pigment will now appear lightly pigmented or unpigmented.

(b) Visual monitoring of melanin production in transformed B 16 cell lines To monitor melanin content of transformed cell lines, cells were trypsinized and transferred to media containing FBS to inhibit trypsin activity. Cells were then counted with a haemocytometer and 2 x 106 cells transferred to a microfuge tube.

Cells were collected by centrifugation at 2,500 rpm for 3 min at room temperature and pellets examined visually.

Five clones transformed with pCMV. TYR. BG12. RYT, namely B16.2 1.11, B16 3.1. 4, B16 3.1. 15, B16 4.12. 2 and B16 4.12. 3, were considerably paler than the B16 controls (Figure 16). Four clones transformed with pCMV. TYR (B16+Tyr 2.3, B16+Tyr 2.9, B16+Tyr 3.3, B16+Tyr 3.7 and B16+Tyr 4.10) and five clones transformed with pCMV. TYR. TYR (B16+TyrTyr 1.1, B16+TyrTyr 2.9, B16+TyrTyr 3.7, B16+TyrTyr 3.13 and B16+TyrTyr 4.4) were also significantly paler than the B16 controls.

(c) Identification of melanin by staining according to Schmorl Specific diagnosis for the presence of cellular melanin can be achieved using a modified Schmorl's melanin staining method (Koss, L. G. (1979). Diagnostic Cytology. J. B. Lippincott, Philadelphia). Using this method, the presence of melanin in the cell is detected by a specific staining procedure that converts melanin to a greenish-black pigment.

Cell populations to be stained were resuspended at a concentration of 500,000 cells per ml in RPMI 1640 medium. Volumes of 200 NI were dropped onto surface- sterilized microscope slides and slides were incubated at 37°C in a humidified atmosphere in 100 mm TC dishes until cells had adhered firmly. The medium was removed and cells were fixed by air drying on a heating block at 37°C for 30 min then post-fixed with 4% w/v paraformaldehyde (Sigma) in PBS for 1 hr. Fixed cells

were hydrated by dipping in 96% v/v ethanol in distilled water, 70% v/v ethanol, 50% v/v ethanol then distilled water. Slides with adherent cells were left for 1 hr in a ferrous sulfate solution [2.5% w/v ferrous sulfate in water] then rinsed in four changes of distilled water, 1 min each. Slides were left for 30 min in a solution of potassium ferricyanide [1% (w/v) potassium ferricyanide in 1 ( (v/v) acetic acid in distilled water]. Slides were dipped in 1% v/v acetic acid (15 dips) then dipped in distilled water (15 dips).

Cells were stained for 1-2 min in a Nuclear Fast Red preparation [0. 1% w/v Nuclear Fast Red (C. I. 60760 Sigma N 8002) dissolved with heating in 5% w/v ammonium sulfate in water]. Fixed and stained cells on slides were washed by dipping in distilled water (15 dips). Cover slips were mounted on slides in glycerol/DABCO [25 mg/ml DABCO (1, 4-diazabicyclo (2.2. 2) octane (Sigma D 2522) ) in 80 % v/v glycerol in PBS]. Cells were examined by bright field microscopy using a 100x oil immersion objective.

The results of staining with Schmorl's stain correlated with the simple visual data illustrated in Figure 8 for all cell lines. When B16 cells were stained with the above procedure, melanin was obvious in most cells. In contrast, fewer cells stained for melanin in the transformed lines B16 2.1. 11, B16 3.1. 4, B16 3.1. 15, B16 4.12. 2, B16 4.12. 3, B16 Tyr 2. 3, B16 Tyr 2. 9, B16 Tyr 4.10, B16 TyrTyr 1. 1, B16 TyrTyr 2.9 and B16 TyrTyr 3.7, consistent with the reduced total tyrosinase activity observed in these cell lines.

(d) Assaying tyrosinase enzyme activity in transformed cell lines Tyrosinase catalyzes the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) and the oxidation of dopa to dopaquinone. Tyrosinase can be measured as its dopa oxidase activity. This assay uses Besthorn's hydrazone (3-methyl-2-benzothiazolinonehydrazone hydrochloride, MBTH) to trap dopaquinone formed by the oxidation of L-dopa.

Presence of a low concentration of N, N'-dimethylformamide in the assay mixture renders the MBTH soluble and the method can be used over a range of pH values.

MBTH reacts with dopaquinone by a Michael addition reaction and forms a dark pink product whose presence is monitored using a spectrophotometer or plate reader. It is assumed that the reaction of the MBTH with dopaquinone is very rapid relative to the enzyme-catalyzed oxidation of L-dopa. The rate of production of the pink pigment can be used as a quantitative measure of enzyme activity (Winder and Harris, 1991; Dutkiewicz et a/., 2000).

B16 cells and transformed B16 cell lines were plated into individual wells of a 96- well plate in triplicate. Constant numbers of cells (25,000) were transferred into individual wells and cells were incubated overnight. Tyrosinase assays were performed as described below after either 24 or 48 hr incubation.

Individual wells were washed with 200 ul PBS and 20 ul of 0.5% v/v Triton X-100 in 50 mM sodium phosphate buffer (pH 6.9) was added to each well. Cell lysis and solubilisation was achieved by freeze-thawing plates at-70°C for 30 min, followed by incubating at room temperature for 25 min and 37°C for 5 min.

Tyrosinase activity was assayed by adding 190 ul freshly-prepared assay buffer (6.3 mM MBTH, 1.1 mM L-dopa, 4% v/v N, N'-dimethylformamide in 48 mM sodium phosphate buffer (pH 7.1)) to each well. Colour formation was monitored at 505 nm in a Tecan plate reader and data collected using X/Scan Software. Readings were taken at constant time intervals and reactions monitored at room temperature, typically 22°C. Results were calculated as the average of enzyme activities as measured for the triplicate samples. Data were analyzed and tyrosinase activity estimated at early time-points when product formation was linear, typically between 2 and 12 min. Results from these experiments are shown below in Tables 1 and 2.

TABLE 1 Cell Line Tyrosinase activity Relative tyrosinase (# OD 505 nm/min/ activity compared to 25, 000 cells) B16-cells (%) B16 0. 0123 100 B16 2. 1. 6 0. 0108 87. 8 B16 2. 1. 11 0. 0007 5. 7 B 16 3. 1. 4 0. 0033 26. 8 B 16 3. 1. 15 0. 0011 8. 9 B 16 4. 12. 2 0. 0013 10. 6 B16 4. 12. 3 0. 0011 8. 9 B 16 Tyr Tyr 1. 1 0. 0043 34 B 16 Tyr Tyr 2. 9 0. 0042 34. 1 B 16 Tyr Tyr 3. 7 0. 0087 70. 7 TABLE 2 Cell Line Tyrosinase activity Relative tyrosinase (# OD 505 activity compared to nm/min/25,000 cells) B16 cells (%) B16 0.0200 100 B16 Tyr 2. 3 0. 0036 18. 2 B 16 Tyr 2. 9 0. 0017 8. 7 B16 Tyr 4. 10 0. 0034 17. 2

These data showed that tyrosinase enzyme activity was inhibited in lines transformed with the constructs pCMV. TYR. BGI2. RYT, pCMV. TYR and pCMV. TYR. TYR 4. Analysis by nuclear transcription run-on assays To detect transcription of the transgene RNAs in the nucleus of B16 cells, nuclear transcription run-on assays were performed on nuclei isolated from actively

dividing cells. The nuclei were obtained according to the cell nuclei isolations protocol set forth in Example 1, above.

Analysis of the nuclear RNA transcripts for the transgene TYR. BG12. RYT from the transfected plasmid pCMV. TYR. BG12. RYT and the endogenous tyrosinase gene is performed according to the nuclear transcription run-on protocol set forth in Example 1, above.

To estimate transcription rates of the endogenous tyrosinase gene in B16 cells and the transformed lines B16 3.1. 4 and B16 Tyr Tyr 1.1, nuclear transcription run- on assays were performed on nuclei isolated from actively dividing cells. The nuclei were obtained according to the cell nuclei isolation protocol set forth in Example 1, above, and run-on transcripts were labelled with biotin and purified using streptavidin capture as outlined in Example 1.

To determine the transcription rate of the endogenous tyrosinase gene in the above cell lines, the amount of biotin-labelled tyrosinase transcripts isolated from nuclear run-on assays was quantified using real time PCR reactions. The relative transcription rates of the endogenous tyrosinase gene were estimated by comparing the levels of biotin-labelled tyrosinase RNA to the levels of a ubiquitously-expressed endogenous transcript, namely murine glyceraldehyde phosphate dehydrogenase (GAPDH).

The levels of expression of both the endogenous tyrosinase and mouse GAPDH genes were determined in duplex PCR reactions. To permit quantitative interpretation of these data, a standard curve was generated using oligo dT- purified RNA isolated from B16 cells. Oligo dT-purification was achieved using Dynabeads mRNA DIRECT Micro Kit according to the manufacturer's instructions (Dynal). Results from these analyses are shown in Table 3.

TABLE 3 Cell Line Tyrosinase and GAPDH RNA Relative levels in biotin-captured nuclear transcription rate of transcription run-on RNAs Tyrosinase gene Ct TYR Ct GAPDH A Ct B16 38.6 27.2 11.5 1.00 B16 3.1. 4 36. 5 24. 4 12. 1 0. 65 B16 TyrTyr 1.1 38.5 26.2 12.4 0.59

These data show clearly that rates of transcription from the endogenous tyrosinase gene in the nuclei of the two silenced B16 cell lines B16 3.1. 4 and B16 TyrTyr 1.1, transformed with pCMV. TYR. BG12. RYT and pCMV. TYR. TYR, respectively, are not significantly different from the rate of transcription from the tyrosinase gene in nuclei of non-transformed B16 cells.

5. Comparison of mRNA in non-transformed and co-suppressed lines Messenger RNA for endogenous tyrosinase and RNA transcribed from the transgene TYR. BG12. RYT are analyzed according to the protocols set forth in Example 1, above.

To obtain accurate estimates of tyrosinase mRNA levels in B16 and transformed lines, real time PCR reactions were employed. Results from these analyses are shown in Table 4.

TABLE 4 Cell Line Tyrosinase and GAPDH mRNA levels Relative levels of in oligo-dT purified total RNAs tyrosinase mRNA Ct TYR Ct GAPDH A Ct B16 33.5 21.9 11.7 1.0 B16 3.1. 4 33. 8 22. 1 11. 7 1. 0 B16 TyrTyr 1. 1 35. 1 23. 0 12. 1 0. i

These data show clearly that the level of tyrosinase mRNA (as poly (A) RNA) in the two silenced B16 cell lines B16 3.1. 4 and B16 TyrTyr 1.1, transformed with pCMV. TYR. BG12. RYT and pCMV. TYR. TYR, respectively, are not significantly different from the level of tyrosinase mRNA in non-transformed B16 cells.

6. Southern analysis Individual transgenic B16 cell lines are analyzed by Southern blot analysis to confirm integration and determine copy number of the transgene. The procedure is carried out according to the protocol set forth in Example 1, above.

EXAMPLE 4 Co-suppression of tyrosinase in Mus musculus strains C57BL/6 and C57BL/6 x DB1 hybrid in vivo 1. Preparation of constructs The interim plasmid TOPO. TYR and test plasmid pCMV. TYR. BG12. RYT (Tyr (hp)) were generated as described in Example 3, above.

2. Generation of transgenic mice Transgenic mice were generated through genetic modification of pronuclei of zygotes. After isolation from oviducts, zygotes were placed on an injection microscope and the transgene, in the form of a purified DNA solution, was injected into the most visible pronucleus (U. S. Patent No. 4,873, 191).

Pseudo-pregnant female mice were generated, to act as"recipient mothers", by induction into a hormonal stage that mimics pregnancy. Injected zygotes were then either cultured overnight in order to assess their viability, or transferred immediately back into the oviducts of pseudo-pregnant recipients. Of 421 injected zygotes, 255 were transferred. Transgenic off-spring resulting from these

injections are called"founders". To determine that the transgene has integrated into the mouse genome, off-spring are genotyped after weaning. Genotyping was carried out by PCR and/or by Southern blot analysis on genomic DNA purified from a tail biopsy.

Founders are then mated to begin establishing transgenic lines. Founders and their offspring are maintained as separate pedigrees, since each pedigree varies in transgene copy number and/or chromosomal location. Therefore, each transgenic mouse generated by pronuclear injection is the founder of a new strain. If the founder is female, some pups from the first letter are analyzed for transgene transmission.

Figures 10 and 11 illustrate the selection of a transformed mouse. As explained in the description of the figures, Figure 10 is a Southern blot from a probe for the plasmid's promoter identifying mouse 038. Figure 11 is a dot blot of that mouse's progeny to identify A037 as transgenic against a control Shiraz 3'.

3. Detection of co-suppression phenotype Visual read-out of successful transgenic mice is an alteration to coat colour. Skin- cell biopsies are harvested from transgenic mice and cultured as primary cultures of melanocytes by standard methods (Bennett et al., 1989; Spanakis et a/., 1992; Sviderskaya et al., 1995).

Melanin pigmentation of transgenic A-generation mouse &num 75-A037 (from founder 038) was visually inspected (see Figure 16) and found to be deficient in broad latero-dorsal areas and in the flanks, resulting from localised downregulation of tyrosinase.

The biopsy area of adult mice is shaved and the skin surface-sterilized with 70% v/v ethanol then rinsed with PBS. The skin biopsy is removed under sterile conditions. Sampling of skin from newborn mice is done after sacrifice of the animal, which is then washed in 70% v/v ethanol and rinsed in PBS. Skin samples are dissected under sterile conditions.

All biopsies are stored in PBS in 6-well plates. To obtain single cell suspensions, PBS is pipetted off and skin samples cut into small pieces (2 x 5 mm) with two scalpels and incubated in 2x trypsin (5 mg/ml) in PBS at 37°C for about 1 hr for newborn samples and up to 15 hr in 1x trypsin (2.5 mg/ml) at 4°C for samples of adult skin (0.5 g in 2.5 ml). This digestion separates epidermis from dermis.

Trypsin is replaced with RPMI 1640 medium to stop enzyme activity. The epidermis of each piece is separated with fine forceps (sterile) and isolated epidermal samples are collected and pooled in 1x trypsin in PBS. Single cell suspensions are prepared by pipetting and separated cells are collected in RPMI 1640 medium. Trypsinization of epidermal samples can be repeated. Pooled epidermal cells are concentrated by gentle centrifugation (1000 rpm for 3 min) and resuspended in growth medium [RPMI 1640 with 5% v/v FBS, 2 mM L-glutamine, 20 units/ml penici)) in, 20 ug/m) streptomycin plus phorbol 12-myristate 13-acetate (PMA) 10 ng/ml (16 nM) and cholera toxin (CTX) 20 ng/ml (1.8 nM) ]. Suspensions are transferred to T25 flasks and incubated without disturbance for 48 hr. Medium is changed and unattached cells removed at 48 hr. After a further 48-72 hr incubation, the medium is discarded, the attached cells washed with PBS and treated with 1x trypsin in PBS. Melanocytes become preferentially detached after this treatment and the detached cells are transferred to fresh medium in new flasks.

Melanocytes in tissue culture are easily distinguishable from keratinocytes by their morphology. Keratinocytes have a round or polygonal shape; melanocytes appear bipolar or polydendritic. Melanocytes may be stained by Schmorl's method (see Example 3, above) to detect melanin granules. In addition, samples of cultures grown on cover slips are investigated by immunofluorescence labelling (see Example 1, above) with a primary murine monoclonal antibody against MART-1 (NeoMarkers MS-614) which is an antigen found in melanosomes. This antibody does not cross-react with cells of epithelial, lymphoid or mesenchymal origin.

4. Analysis by nuclear transcription run-on assays To detect transcription of the tyrosinase endogenous gene and transgene RNAs in the nucleus of primary culture melanocytes, nuclear transcription run-on assays are performed on cell-free nuclei isolated from actively dividing cells, according to the cell nuclei isolation protocol set forth in Example 1, above.

Analysis of nuclear RNA transcripts for the tyrosinase endogenous gene and the transgene from the transfected plasmid pCMV. TYR. BGi2. RYT are performed according to the nuclear transcription run-on protocol set forth in Example 1, above.

5. Comparison of mRNA in non-transformed and co-suppressed lines Messenger RNA for endogenous tyrosinase and RNA transcribed from the transgene TYR. BG12. RYT are analyzed according to the protocols set forth in Example 1, above.

6. Southern analysis Primary culture melanocytes are analyzed by Southern blot analysis to confirm integration and determine copy number of the transgene. This is carried out according to the protocol set forth in Example 1, above.

EXAMPLE 5 Co-suppression of cz-1, 3,-galactosyl transferase (GalT) in Mus musculus strain C57BL/6 in vivo 1. Preparation of genetic constructs (a) Plasmid TOPO. GALT Total RNA was purified from cultured murine 2. 3D17 neural cells and cDNA prepared as described in Example 2.

To amplify the 3'-UTR of the murine a-1, 3,-galactosyl transferase (GaIT) gene, 2 uf of this mixture was used as a substrate for PCR amplification using the primers: GALT-F2: CAC AGA CAG ATC TCT TCA GG and GALT-R1 : ACT TTA GAC GGA TCC AGC AC.

The PCR amplification was performed using HotStarTaq DNA polymerase according to the manufacturer's protocol (Qiagen). PCR amplification conditions involved an initial activation step at 95°C for 15 mins, followed by 35 amplification cycles of 94°C for 30 secs, 55°C for 30 secs and 72°C for 60 secs, with a final elongation step at 72°C for 4 mins.

The PCR amplified region of GaIT was column purified (PCR purification column, Qiagen) and then cloned into pCR2.1-TOPO according to the manufacturer's instructions (Invitrogen), to make plasmid TOPO. GALT.

(b) Test plasmid Plasmid pCMV. GALT. BG12. TLAG Plasmid pCMV. GALT. BG12. TLAG (GaIT (hp), Figure 9) contains an inverted repeat, or palindrome, of a region of the Murine 3'UTR GaIT gene that is interrupted by the insertion of the human p-globin intron 2 sequence therein. Plasmid pCMV. GALT. BG12. TLAG was constructed in successive steps: (i) the GALT sequence from plasmid TOPO. GALT was sub-cloned in the sense orientation as a Bgill-to-BamHI fragment into Bg/ll-digested pCMV. BGI2 to make plasmid pCMV. GALT. BGl2, and (ii) the GALT sequence from plasmid TOPO. GALT was sub-cloned in the antisense orientation as a Bglll-to-BamHl fragment into BamHl- digested pCMV. GALT. BG ! 2 to make plasmid pCMV. GALT. BG12. TLAG.

2. Generation of transgenic mice Transgenic mice were generated through genetic modification of pronuclei of zygotes. After isolation from oviducts, zygotes were placed on an injection microscope and the transgene, in the form of a purified DNA solution, was injected into the most visible pronucleus (US patent number: 4, 873, 191).

Pseudo-pregnant female mice were generated, to act as"recipient mothers", by induction into a hormonal stage that mimics pregnancy. Injected zygotes were then either cultured overnight in order to assess their viability, or transferred immediately back into the oviduct of pseudo-pregnant recipients. Of 99 injected zygotes, 25 were transferred. Transgenic off-spring resulting from these injections are called"founders". To determine that the transgene has integrated into the mouse genome, off-spring are genotyped after weaning. Genotyping was carried out by PCR and/or by Southern blot analysis on genomic DNA purified from a tail biopsy.

Founders are then mated to begin establishing transgenic lines. Founders and their offspring are maintained as separate pedigrees, since each pedigree varies

in transgene copy number and/or chromosomal location. Therefore, each transgenic mouse generated by pronuclear injection is the founder of a new strain.

If the founder is female, some pups from the first letter are analyzed for transgene transmission.

3. Detection of co-suppression phenotype The enzyme a-1, 3,-galactosyl transferase (GaIT) catalyzes the addition of galactosyl sugar residues to cell surface proteins in cells of all mammals except humans and other primates. The epitope enabled by the action of GaIT is the predominant antigen responsible for the rejection of xenotransplants in humans.

Cytological analyses of GaIT expression levels in peripheral blood leukocytes (PBL) and splenocytes using FACS confirms the down regulation of the gene's activity.

Analysis of Peripheral Blood Leukocyfies and Splenocytes from transaenic mice by FACS To analyze cells from transgenic mice transformed with the GaIT construct, FACS assays on peripheral blood leukocytes (PBL) and splenocytes are undertaken.

White blood cells are the most convenient source of tissue for analysis and these can be isolated from either PBL or splenocytes. To isolate PBL, mice are bled from an eye and 50 to 100 pl of blood collected into heparinized tubes. The red blood cells (RBCs) are lysed by treatment with NH4CI buffer (0. 168M) to recover the PBLs.

To obtain splenocytes, animals are euthanased, the spleens removed and macerated and RBCs lysed as above. The generated splenocytes are cultured in vitro in the presence of interleukin-2 (IL-2 ; Sigma) to generate short term T cell cultures. The cells are then fixed in 4% w/v PFA in PBS. All steps are performed on ice. GaIT activity can be most conveniently assayed using a plant lectin (IB4 ; Sigma), which binds specifically to galactosyl residues on cell surface proteins.

GaIT is detected on the cell surface by binding IB4 conjugated to biotin. The

leukocytes are then treated with streptavidin conjugated to Cy5 fluorophore.

Another cell marker, the T cell specific glycoprotein Thy-1, is labelled with a fluorescein isothiocyanate-conjugated antibody (FITC ; Sigma). The leukocytes are incubated in a mixture of the reagents for 30 min to label the cells. After washing, the cells are analyzed on the FACScan. (Tearle, R. G. et al., 1996).

4. Analysis by nuclear transcription run-on assays To detect transcription of transgene RNAs in the nucleus of splenocytes, nuclear transcription run-on assays are performed on cell-free nuclei isolated from actively dividing cells. In vitro culturing of splenocytes in the presence of IL-2 generates short term T cell cultures. The nuclei are obtained according to the cell nuclei isolation protocol for suspension cell cultures, set forth in Example 1 above.

Analysis of nuclear RNA transcripts for the GaIT endogenous gene and the transgene from the transfected plasmid pCMV. GALT. BG12. TLAG is performed according to the nuclear transcription run-on protocol set forth in Example 1, above.

5. Comparison of mRNA in non-fransformed and co-suppressed lines Messenger RNA for endogenous GaIT and RNA transcribed from the transgene GALT. BG12. TLAG are analyzed according to the protocols set forth in Example 1, above.

6. Southern analysis Individual transgenic splenocyte cell lines are analyzed by Southern blot analysis to confirm integration and determine copy number of the transgenes. This is carried out according to the protocol set forth in Example 1, above.

Figures 12 to 15, and the descriptions of these figures given above, further illustrate the methods relating to these transformed mice.

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