AU757433B2 - Impaired BRCA2 function in cells and non-human transgenic animals - Google Patents

Description

WO 99/10479 PCT/US98/17566 IMPAIRED BRCA2 FUNCTION IN CELLS AND NON-HUMAN TRANSGENIC

ANIMALS

FIELD OF THE INVENTION The present invention relates to cells and non-human transgenic animals that have been engineered to incorporate a Brca2 gene (GenBank Accession No. U65594) that has an impaired ability to associate, either directly or indirectly, with Rad51. In particular, Brca2 activity was reduced in cells by targeted disruption of the Brca2 gene such that the domain that codes for the Rad51, or Rad51 complex, interacting region is removed but the remainder of the coding sequence is left intact and is expressed. The engineered cells were subsequently used to generate transgenic animals that produced the altered Brca2 protein.

BACKGROUND OF THE INVENTION Cellular DNA normally exists in a dynamic environment.

Cellular functions of repair, recombination, replication and 2cell cycle regulation are intimately interwoven to maintain genomic stability and generate genetic diversity (reviewed by Petes et al., 1991, Recombination in yeast, In: Molecular and Cellular Biology of the Yeast Saccharomyces (eds. J. R.

Boach, J. R. Pringle, and E. W. Jones), pp. 407-521, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Drapkin et al., 1994, Cell, 77:9-12; Kuhn et al., 1995, Genes Dev. 9:193-203; Friedberg, et 1995, DNA repair and mutagenesis, pp. 147-192, ASM Press Washington, D. Li, et al., 1995, Cell 83:1079-1089). A mutation in a gene whose product is critical to any of these processes may result in a variety of clinical signs that include neurological disorders, immunodeficiency, and predisposition to cancer.

Understanding the molecular mechanisms of repair and recombination will be beneficial to understanding the etiology of disease caused by defects in these processes.

The mouse is ideal for studying the dynamic nature of DNA.

Similarities in human and mouse genomic constitution, 1 WO 99/10479 PCT/US98/17566 including intron-exon boundaries and the position of regulatory elements, as well as the spatial transcriptional regulation of homologous genes is remarkable (Lyon and Searle, 1989, Genetic variants and strains of the laboratory mouse, 2nd ed. Oxford University Press, Oxford). In addition, anatomical similarities between mice and humans provide the opportunity for direct physiological comparisons.

Targeted disruption of genes encoding protein products such as the p53 tumor suppressor (Donehower et al., 1992, Nature 356:215-221), the mismatch repair proteins (Baker et al., 1995, Cell 82:309-319; de Wind et al., 1995, Cell 82:321-330) and the xeroderma pigmentosa complementation groups (Sands et al., 1995, Nature 377:169-173; de Vries et al., 1995, Nature 377:169-173; Nakane et al., 1995, Nature 377:165-168) have revealed striking similarities to inherited disorders in humans.

A number of different DNA repair pathways are responsible for correcting a variety of specific DNA lesions.

These pathways include nucleotide excision repair, mismatch repair and double-strand break (DSB) repair. The mechanisms responsible for nucleotide excision repair and mismatch repair are fairly well understood, and mutations affecting these processes have been characterized (reviewed in Friedberg, 1992, Cell 71:887-889; Cleaver, 1994, Cell 76:1- However, the mechanisms responsible for the repair of DSBs remain poorly understood. Several inherited disorders of mammals feature defects in the repair of DSBs that are associated with hypersensitivity to ionizing radiation and immunodeficiency. These include Ataxia-Telangiectasia (AT) in humans (reviewed by Lehmann and Carr, 1995, Trends in Genet. 11:375-377) and autosomal recessive scid (severe combined immunodeficiency) in mice (Roth et al., 1992, Cell 70:983-991) and in horses (Wiler, et al., 1995, Proc. Natl.

Acad. Sci. 92:11485-11489).

ScRad51 is a member of the RAD52 epistasis group in Saccharomyces cerevisiae, and is a major component in the yeast DSB repair pathway (by homologous recombination); a 2 -3pathway called recombinational repair (reviewed by Friedberg, et 1995, DNA repair and mutagenesis, pp. 523-594, ASM Press Washington, This pathway repairs genetic damage caused by ionizing radiation. The mouse homologue of ScRad51, MmRad51, appears to have a similar function (Shinohara et al., 1993, Nature Genet. 4:239-243; Lim and Hasty 1996, Mol.

Cell. Biol. 16:7133-7143); however, the precise mechanism of action is not well understood. For ScRad51, protein:protein associations are critical for function.

Consequently, a yeast two-hybrid system was used to isolate proteins that associate with MmRad51 to better understand recombinational repair in mammalian cells, and mouse Brca2 was isolated (Sharan et al., 1997, Nature, 386 804-810). A phenotypic comparison between MmRad51 and Brca2deficient embryos and cells suggest that a protein:protein association is important for their function. Similar to MmRad51, Brca2 function is critical for early embryonic development, cell proliferation or viability and the repair of y radiation induced damage.

People with mutations in Brca2 are predisposed to breast cancer (Wooster, et al., 1994, Nature 265: 2088-2090 Smith et al., Nature Genet. 2: 128-131 Easton, et al., 1993, A. J. Hum. Genet. 52: 678-701). Neoplasia is associated with loss of heterozygosity of the non-mutated allele in tumors, suggesting Brca2 is a tumor suppressor. Brca2 is a 3,418 amino acid protein with no significant homology to any other genes (Wooster, et al., 1995, Nature 378: 789-792; Tavtigan, et 1996, Nature Genet. 13: 120-122). The mouse Brca2 protein is 3,328 amino acids and the overall identity is 58% between mouse and human Brca2 (Sharan, et al., 1997, Genomics 40: 234-241).

41 6 25 The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

SUMMARY OF THE INVENTION Brca2 is a tumor suppressor and mediates Rad51 function.

I Consequently, it is possible that absence of Brca2 destabilizes or reduces 23 January 2001 -4- Rad51 function which in turn is mutagenic. Some of these mutations could promote cancer. Mice and cells have been generated with subtle mutations that inhibit the direct or indirect association of Brca2 with Mouse Rad5l Data described here demonstrate that a subtle mutation which removes only a small portion of Brca2 that associates, either directly or indirectly, with MmRad51 exhibit a phenotype that suggests partial function. These brca2mutant cells are viable yet hypersensitive to ionizing radiation suggesting they are deficient in the repair of double strand breaks in DNA. In addition, embryonic fibroblasts undergo premature replicative senescence, similar to cells deficient for the Ku autoantigen (United States Patent Application Serial No. 08/695,866, filed August 8, 1996), another protein involved in the repair of double strand breaks.

An aspect of the present invention is to provide animal cells which express an altered form of Brca2 that is impaired for its ability to associate with MmRad51, either directly of indirectly.

In one aspect the present invention provides a non-human diploid animal cell containing an engineered mutation in at least one allele of the Brca2 gene located in a region of the Brca2 gene downstream from nucleic acid residue 9265 of GenBank Accession No. U65594 and wherein the mutation produces a o 20 protein that is impaired for its ability to directly or indirectly associate with MmRad51.

01"0 In another aspect the present invention provides a non-human diploid %0:0 00 animal cell containing a first engineered mutation in one allele of the Brca2 gene and a second mutation engineered in the other allele of the Brca2 gene wherein the mutations produce a protein that is impaired for its ability to directly or indirectly associate with l •An additional aspect of the present invention is to provide mammalian, preferably mouse, embryos or mammals, preferably mice, which express an altered form of Brca2 that has an impaired ability to associate with MmRad51, either directly or indirectly.

In one aspect the present invention provides a non-human transgenic _fRA 'Vlwhich comprises an engineered mutation which decreases the 4aexpression or alters function of at least one allele of the Brca2 gene, wherein said mutation is located in a region of the Brca2 gene downstream from nucleic acid residue 9265 of GenBank Accession No. U65594 and wherein the mutation produces a protein that is impaired for its ability to directly or indirectly associate with MmRad51.

In another aspect the present invention provides a method for identifying a gene, which in a mutant form rescues the premature replicative senescent phenotype of cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad5l, comprising the steps of: introducing at least one genetic mutation into the cells; measuring proliferation ability of the mutated cells using 3T3, 3T9, or population doubling analysis; isolating the mutated cells that are not senescent after passage 8; and identifying the gene in the cells which has been mutated in step In another aspect the present invention provides a method for identifying a compound that rescues cells containing an engineered mutation in at least one allele of the Brca2 gene from premature replicative senescence, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad5, comprising the steps of: contacting the cells with a compound; and measuring the proliferation ability of the cells using 3T3, 3T9, or population doubling analysis; whereby the presence of mutated cells that are not senescent after passage 8 identifies the compound.

In another aspect the present invention provides a method for screening for compounds or molecules that increase the incidence of cancer in animals having cells containing an engineered mutation in at least one allele of the 4b- Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, comprising the step of exposing the animals to said compounds and identifying a compound or molecule that increases the incidence of cancer in the animals; In another aspect the present invention provides a method for screening for compounds or molecules that decrease the incidence of cancer in animals having cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad5l, comprising the step of exposing the animals to said compounds and identifying a compound or molecule that decreases the incidence of cancer in the animals.

These and other aspects of the present invention which will be apparent from the detailed description of the invention are exemplified by a mouse cell containing two chromosomal alleles of the Brca2 gene, wherein at least one of said alleles contains a mutation that produces Brca2 having an impaired ability to directly or indirectly associate with MmRad51.

Another embodiment of the present invention, is a mutant mouse embryo which produces Brca2 that has been engineered to have an impaired ability to directly or indirectly associate with Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

WO 99/10479 PCT/US98/17566 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Targeting strategy for Brca2 locus. A.

Deletion of exon 27 (coding nucleotides 9420-9984) with targeting vector pMB2TVhprt. This targeted allele is called brca2 l ex An HPRT selection cassette was flanked upstream by a 5.4 kb Apal/Smal genomic Brca2 fragment and downstream by a 1.9 kb HindIII/Smal genomic fragment; thus, creating a 2.5 kb deletion that removes all of exon 27 of Brca2. Positive selection, HPRT minigene; negative selection, thymidine kinase (tk) cassette; plasmid backbone (pKS, Stratagene), wavy line. Southern analysis is an BglII digest B. Deletion of most of exon 26 and all of exon 27 (coding nucleotides 9265-9984) with targeting vector pMB2TVneo. This allele is called brca2l ex 2 A neomycin phosphotransferase (neo) selection cassette was flanked upstream by a 4.6 kb Apal/Clal genomic Brca2 fragment and downstream by a 1.9 kb HindIII/Smal genomic fragment; thus, creating a 3.3 kb deletion that removes most of exon 26 and all of exon 27 of Brca2. Positive selection, neo cassette; negative selection, thymidine kinase (tk) cassette; plasmid backbone (pKS, Stratagene), wavy line.

Southern analysis is an BglII digest.

Figure 2. Exposure of control and brca21ex l /brca2 1e cells to genotoxic agents. Survival fractions (100% X number of colonies after exposure to genotoxic agent number of colonies not exposed to genotoxic agent) were measured after 10,000 cells were plated onto a 10 cm plate and colonies counted 10 days later. A. Dose response curve to 7radiation. Controls are wild-type Hprt positive cells (AB1, one clone), wild-type Hprt negative cells (AB2.2, three clones), brca2 e cells (eight clones) and brca21 2 (six clones). Each of these groups of control clones resulted in the same survival fraction and are averaged for this curve.

Eight clones of brca21ex l /brca21e' x cells were averaged. B.

Dose response curve to ultraviolet light. The average of three brca2'"1/+ clones and two brca2 e x2 clones are 5 WO 99/10479 PCTIUS98/17566 presented for controls. The average of five brca2 1 ex l /brca2 ex 2 clones are presented.

Figure 3. Growth characteristics of brca2Iexl/brca2 lex2 embryonic fibroblasts. Mouse embryonic fibroblasts (MEF) were isolated from wild-type E15.5 day 129SvEv embryos and brca2exl/brca2 l ex2 MEF were isolated from E15.5 day chimeric embryos (129SvEv cells injected into Swiss Webster blastocyts) brca21e"/brca21ex 2 MEF were isolated from embryos with black eyes by selection in 90 mM G418 for 10 days.

brca21exl/brca2le x2 MEF were maintained with and without G418 selection for all experiments (presence or absence of G418 did not affect growth). All experiments begin with passage 1 cells. MEF were grown in M10 (10% fetal calf serum from HyClone, Dulbecco's Modified Eagle's Medium from GibcoBRL, 2 mM L-Glutamine, 49.5 U/ml Penicillin and 38.8 Ag/ml Streptomycin). A. Growth curve. 8 X 104 MEF were plated onto eight 3.5 cm plates and individual wells of cells were trypsinized and counted over 11 days. B. Percentage of cells in S phase. MEF (4 X 1 0 5 .were grown on a 6 cm plate for 2 days. MEF were continuously exposed to 10 AM 5-bromo-2'deoxyuridine (BrdU) and harvested over a 48 hour time course.

Cells were permeabilized and exposed to fluorescently labeled anti-BrdU antibodies as well as propidium iodide to stain DNA. A fluorescence activated sorter (FACS) analysis was performed on the cells (2 X 10 5 cells) to determine the percentage of cells that had incorporated BrdU (indicating DNA synthesis and thus cell cycle progression) at each time point. C. Colony formation at low density plating. For each clone, 5000 MEF were plated onto a 10 cm plates (three plates for each clone) and grown for 14 days. The colonies were stained with crystal violet and the number of colonies were counted. Colonies are >3 cells. D. Colony size distribution (CSD). The percentage of colonies with >15 cells are compared to the total number of colonies with >3 cells. E.

Measurement of life span. The life span was determined by measuring the number of passages the MEF could undergo before proliferation stopped. MEF were plated onto three 6 cm 6 WO 99/10479 PCT/US98/17566 plates (1 X 10 5 cells/ plate). MEF were trypsinized every days and the total number of cells counted. MEF were then plated back onto three 6 cm plates and the procedure continued until there was not enough MEF to plate onto three plates. Then 1 X 10 5 MEF were plated onto only two plates and finally only one plate. MEF were considered to be completely senescent when fewer than 1 X 10 5 cells remained. One clone of wild-types cells spontaneously immortalized.

5.0. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the production of Brca2-impaired cells, and Brca2-impaired non-human animals.

The non-human transgenic animals contemplated by the present invention generally include any vertebrates, and preferably mammals, which encode a Brca2 homolog. Such non-human transgenic animals may include, for example, transgenic pigs, transgenic rats, transgenic rabbits, transgenic cattle, transgenic goats, and other transgenic animal species, particularly mammalian species, known in the art.

Additionally, bovine, ovine and porcine species, other members of the rodent family, e.g. rat, as well as rabbit and guinea pig and non-human primates, such as chimpanzee, may be used to practice the present invention. Particularly preferred animals are rats, rabbits, guinea pigs, and most preferably mice.

Given the apparent similarity between the yeast and murine DSB repair mechanisms, the murine Brca2 sequence utilized herein can be used as a heterologous probe to identify and isolate the corresponding genes from any of a wide variety of animal species. Typically, hybridization conditions are adjusted in accordance with the relatedness of the probe and target sequences. For example, hybridization/washing conditions should be of a lower stringency when the cDNA library (or target sequence) is derived from an organism different from the type of organism from which the labeled sequence was derived. With respect to the cloning of a Brca2 homolog, using murine Brca2 probes, 7 WO 99/10479 PCT/US98/17566 for example, hybridization can, for example, be performed at 0 C overnight in Church's buffer SDS, 250 mM NaHP04, 2AM EDTA, 1% BSA). Washes can be done with 2XSSC, 0.1% SDS at 650C and then at 0.1XSSC, 0.1% SDS at 650C.

Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Alternatively, the labeled Brca2 nucleotide probe may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.

Further, a Brca2 gene homolog may be isolated from nucleic acid of the organism of interest by performing PCR using two oligonucleotides designed from the Brca2 sequences utilized herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue, such as.

choroid plexus, known or suspected to express a Brca2 gene allele.

The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an Brca2 gene. The PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library.

Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source one known, or suspected, to express the obR gene, such as, for example, choroid plexus or brain 8 WO 99/10479 PCT/US98/17566 tissue). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer.specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies which may be used, see Sambrook et al., 1989, supra Although virtually any animal cells may be utilized to practice the present invention, preferred embodiments of the present invention include diploid mouse cells, mouse embryos and mice that contain two chromosomal alleles of the Brca2 gene, wherein at least one of the Brca2, alleles contains a mutation such said cell produces some Brca2 protein that is impaired with its function to associate, either directly or indirectly, with MmRad51. Such Brca2-impaired cells and mice are deemed to be useful as, inter alia, disease models for the analysis and testing of therapeutic agents, and the effects of mutagenic stimuli such as radiation and chemical mutagens.

Replicative or cellular senescence is a process common to cells that leads to their terminal arrest and probably functions as a control against tumor formation and may reflect organismal aging (Campisi 1996, Cell 84:497-500).

Given that Brca2-impaired cells, and possibly animals, exhibit features of accelerated senescence, the presently described cells and animals are also deemed to be useful for the study of biological aging, and agents for retarding the same.

In particular, methods are contemplated for the screening for compounds, conditions, or compensatory mutations, that partially or fully rescue the proliferation and or senescence abnormalities associated with Brca2impaired cells. Examples of such conditions include, but are 9- WO 99/10479 PCT/US98/17566 not limited to, the over expression of transfected genes of endogenous genes, or the mutagenesis of genes and the like.

Examples of such compounds include peptides, peptides analogues, antisense or aptameric oligonucleotides, organic molecules, including prostaglandins, and the like.

As discussed above, one embodiment of the present invention includes a mouse cell containing two chromosomal alleles of the Brca2 gene, wherein at least one of said alleles contains a mutation such that said cell produces Brca2 having an impaired ability to associate with MmRad51.

Additional embodiments of the present invention include nonhuman animal embryos, and non-human transgenic animals incorporating the Brca2-impaired cells.

As used herein,"brca2-impaired" means that at least one of the two wild-type Brca2 chromosomal alleles has been mutated to encode Brca2 having an impaired ability to directly or indirectly associate with MmRad51 or any other protein that associates with MmRad51. Brca2-impaired products can be easily measured using standard molecular biology techniques. For example, one can measure altered Brca2 messenger RNA levels by using reverse transcriptase polymerase chain reaction (RT-PCR) (see Figure Thus, the term brca2-impaired also includes homozygous, as well as a heterozygous genotypes, although a homozygous genotype is preferable.

The mutation in the Brca2 gene is preferably a deletion mutation that removes part or all of the nucleotides that codes for the domain that mediates an association, either direct or indirect, with MmRad51, although substitution mutations, frame shift mutations, and/or insertion mutations are included within the scope of the present invention.

Substitution mutations can be prepared by site directed mutagenesis, as described by Hasty et al., 1991, Nature 350:243-246, so as to introduce a stop codon or other mutation near the region that codes for the domain that associates with MmRad51, either directly or indirectly so as to give rise to a truncated Brca2 protein product having an 10 WO 99/10479 PCT/US98/17566 impaired ability to directly or indirectly associate with MmRad51. Similarly, insertion mutations can be introduced within the Brca2 gene taking advantage of the convenient restriction sites therein, such as any of the exonic restrictions sites or other sites which are easily identified by exonic sequencing of the Brca2 gene and restriction mapping (Figure and the techniques described by Hasty et al., 1991; Joyner et al., 1989. Another method of introducing an insertion or other mutation consists of infecting with a retrovirus which integrates in the Brca2 locus, thereby creating a mutated brca2 allele as described by von Melchner et al., Genes and Dev 6:919-927. However, the mutants of the present invention preferably lack part of the DNA sequence coding for Brca2 so that a defective brca2 allele is more likely made such that the produced Brca2 protein is impaired in its ability to directly or indirectly associate with MmRad51.

The coding region of the Brca2 gene is approximately 9984 bp in size. For the purposes of this present invention, the nucleotides encoding the Brca2 gene shall be numbered according to the gene bank accession U65594. Deletion mutants can be produced by eliminating a DNA fragment from a coding region of the Brca2 gene so that proper folding or substrate binding of the Brca2 protein with MmRad51 or another protein in this complex is impaired. The size of the deletion may vary. Alternatively, deleting a single base pair or two base pairs or any number of base pairs from the coding region would could result in impaired activity. In the latter instance, a truncated polypeptide may be produced because polypeptide synthesis is aborted due to a frame shift-induced stop codon. For a general review of mutagenesis and mutation see "An Introduction to Genetic Analysis", 4th edition, 1989 Suzuki, A. Griffiths, J.

Miller, and R. Lewontin, eds.), W. H. Freeman Co., N. Y., New York.

Still, changing a single base pair in the coding region of the brca2 gene could also be a mutation which, if 11 WO 99/10479 PCT/US98/17566 resulting in an amino acid change, could alter the proper folding of the Brca2 protein and thereby create an Brca2 impaired activity. A single amino acid change so generated could also alter the affinity of Brca2 for its substrate and thereby result in impaired association with MmRad51 or another protein in this complex. Another alternative would be to generate a deletion or other mutation in the non-coding region of the Brca2 gene which affected the proper splicing of the Brca2 messenger RNA. Such a mutation could effectively create a mutant Brca2 transcript which was missing an entire exon or several exons as compared to the wild type Brca2 message. Another alternative is to delete a non-coding regulatory region to decrease expression of the Brca2 gene. Alternatively, promoter sequences could be deleted or altered that would diminish transcription of the Brca2 gene and reduced transcription could result in an insufficiency of protein such that Brca2 association with MmRad51, either directly or indirectly, is impaired.

It is also possible to alter the expression of a given gene by altering codon usage in the gene. Alterations of this sort preserve the amino acid sequence of the product while increasing or decreasing the levels of expression.

Antisense RNA transgenes may also be employed to partially or totally knock-out expression of specific genes (Helene., C. and Toulme, 1990, Biochimica Bioshys. Acta 1049:99; Pepin et al., 1991 Nature 355:725; Stout, J. and Caskey, 1990, Somat. Cell Mol. Genet. 16:369; Munir et al., 1990 Somat Cell Mol. Genet. 16:383, each of which is herein incorporated herein by reference).

"Antisense polynucleotides" are polynucleotides that: are complementary to all or part of a reference target sequence, such as the sequence of Brca2 gene, and specifically hybridize to a complementary target sequence, such as a chromosomal gene locus mRNA. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions or transpositions, so long as specific hybridization to the relevant target 12 WO 99/10479 PCT/US98/17566 sequence is retained as a functional property of the polynucleotide. Complementary antisense polynucleotides include antisense which can hybridize specifically to individual mRNA species and hinder or prevent transcription or RNA processing of the mRNA species and/or translation of the encoded polypeptide (Ching et al., 1989, Proc. Natl.

Acad. Sci. USA 86:10006-10010; Broder et al., Ann. Int. Med.

113:604-618; Loreau et al., 1990, FEBS Letters 274:53-56; Holcenberg et al., W091/11535; W091/09865; W091/04753; W090/13641; and EP 386563, each of which is incorporated herein by reference). An antisense sequence is a polynucleotide sequence of at least about 15 contiguous nucleotides in length, typically at least 20 to nucleotides in length, and preferably more than nucleotides in length that is substantially complementary to nucleotides to a target gene sequence, or sequences in a cell. In some embodiments, antisense sequences may have substitutions, additions, or deletions as compared to the complementary target sequence but as long as specific hybridization is retained, the polynucleotide will generally function as an antisense inhibitor of gene expression.

For the purposes of he present invention, the antisense sequence is complementary to an endogenous Brca2 target gene sequence. In some cases, sense sequences corresponding to the brca2 target region may function to suppress expression, particularly by interfering with transcription.

Alternatively, an antisense polynucleotide will generally suppress Brca2 expression at a post transcriptional level.

Given that antisense polynucleotides inhibit the production of polypeptide(s) in cells, they may further alter a non-human transgenic animal's capacity to produce Brca2.

Antisense polynucleotides may be produced from a heterologous expression cassette inserted into transgenic pluripotent embryonic stem cells which may subsequently be used to generate the presently described Brca2-impaired animals.

13 WO 99/10479 PCT/US98/17566 The gene modified animal cells of the present inventions can be prepared by any of several techniques that are well established in the art. In particular, techniques conceptually similar to those taught in U. S. Patent No.

5,464,764 issued to Capecchi nd Thomas on November 7, 1995, herein incorporated by reference, may be used. In general, Brca2-impaired cells may be engineered using the following steps: Constructing a targeting vector comprising a cloning vector and a DNA fragment containing at least one positively selectable marker gene (positive selection marker), flanked by two non contiguous regions of the mouse Brca2 gene or genomic locus which are in the same 5' to 3' orientation to one another referred to as the regions of homology; Included in the targeting vector a negatively selectable marker gene (negative selection marker) adjacent to one of the regions of homology. This negatively selectable marker may increase the likelihood of recovering the desired homologous recombination event deleting a portion of the Brca2 gene but it is not required; Transfecting wild-type Brca2 mouse cells with the targeting vector of step Screening or selecting for said marker(s) in the resulting transfected mouse cells of step and Screening for Brca2-impaired mouse cells from those cells in step which are found to contain or express said positive selection marker(s) and not express said negative selection marker(s) The precise Brca2 gene or gene locus sequences which must be present in the targeting vector of step will depend on the sequences chosen for the deletion, and the restriction nucleases to be employed in the engineering of the deletion mutant.

The specific regions of homology required in step (1) depend on the specifics of the deletion in the targeting vector. In general, the size of the homology regions used in the targeting vector will be at least about 400 bp, though 14 WO 99/10479 PCT/US98/17566 longer or shorter regions could be used. In general it is preferable to use homology regions of approximately 1.5 kb or greater to insure a high degree of targeting efficiency. The targeting vector described in detail in Figure 1, the 5' and 3' homology regions on both sides of the deletion were greater than 1.5 kb.

The size of the deletion may also vary and depends on the regions of homology used in the targeting vector. That is, since non-contiguous regions of homology are used in the targeting vector, that region in the wild-type allele which is located between the regions of homology constitutes the region to be deleted upon homologous recombination with the targeting vector. The region to be deleted in the present invention is approximately 2.5 kb for brca2'exI and 3.3 kb for brca2lex2; however, the exact size is not critical and either more or less could be deleted from the locus and still result in brca2-deficiency. It is preferable that the deletion include at least one exon or a portion of an exon of the Brca2 gene so as to result in mutant brca2 messenger RNA.

The particular positive and negative selection markers employed in the present invention are not critical thereto.

Examples of preferred positive and negative selection markers are listed in Table 1. The positive selectable marker should be located between the regions of homology and the negative marker, if one is used, should be outside the regions of homology, either 5' or 3' to those regions as shown in Figure la and lb. The regions of homology should be in the same to 3' orientation to one another while the orientation of the positive and negative selectable markers are not critical.

It is not critical to include a negative selectable marker, though this may increase the efficiency of targeting.

The positive selectable marker should be engineered to be functional in the transformed cells in which the gene targeting is being performed. Positive and/or negative selection markers are functional in the transfected cells if the phenotype expressed by the DNA sequences encoding such selection markers is capable of conferring either a positive 15 WO 99/10479 PCT/US98/17566 or negative selection characteristic for the cell that is expressing the sequence. The means by which the positive selectable marker gene is made functional is not critical to the present invention. Positive selection is accomplished by exposing the cells to an appropriate agent which kills or otherwise selects against cell not containing an integrated positive selection marker. Examples of such agents are listed in Table 1. The positive selectable marker gene may have a promoter driving its expression or it may be driven by the juxtaposition of transcriptional elements at the target locus with the positive selectable marker. This latter method requires that those transcriptional elements are active in the transformed cells.

The mutation engineered in the targeting vector can contain DNA sequences between the regions of Brca2 gene homology in addition to a positive selection marker, an oligonucleotide linker, in place of the deleted Brca2 DNA.

The oligonucleotide linker is generally 8-10 nucleotides in length, but can be longer, e.g. about 50 nucleotides, or shorter, e.g. 4, 5 or 7 nucleotides. The preferred length of the oligonucleotide linker is about 20 to 40 nucleotides in length. The DNA sequence of the oligonucleotide linker is not critical.

The method of inserting the oligonucleotide between the regions of homology in the targeting vector DNA will depend upon the type of oligonucleotide linker used. Palindromic double stranded linkers containing one or more restriction nuclease sites in the oligonucleotide sequence (New England Biolabs) may be inserted by well known procedures (Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N. pMB9, pBR325, pKH47 (Bethesda Research Laboratories), Oligonucleotide linkers may also be inserted into deletions in plasmid DNA by tailing ends with complementary homopolymers using terminal transferase (Maniatis et al., supra). Alternatively, an oligonucleotide linker may be inserted into a deletion in a plasmid by bridging, through annealing of 16 TABLE 1 Selectable Markers for use in Gene Targeting Gene neo hyg hisD gpt hpr t IUSV-tk

T

Selective M'e Agents G4 18 4+ Ilygromycin Histidinol Xanthine Iypoxanthine Gancyclovir

FIAU

6-thioguanine 6-thioxanthine None None 5-fluorocytosine Preferred concentration of Selective Agent 50 1000 pig/mi 10 1000 pg/mi 5 500 pg/mi 50-500 gg/mi 0.01 10 M 0.05 200 pM 0.02 100 pM 0.1 100 pg/mi 0.1 100 pg/mi None None 10 500 pg/mi organism Eukaryotes Eukaryotes Animals Animals All Animals Animals All Animals Animals Animals All hprt gpt Diptheria, toxin Ricin Toxin cytosine deaminase WO 99/10479 PCT/US98/17566 oligonucleotides containing ends complementary to a cleaved plasmid's 3'-recessed and 3'-protruding cohesive ends, followed by filling in of the gap complementary to the oligonucleotide sequence with DNA polymerase Klenow fragment). After subsequent ligation with T4 DNA ligase, closed circular DNA molecules can be regenerated. If the targeting vector is designed such that the deleted region interrupts an exon, by the judicious choice of oligonucleotide linker length and sequence, frame shift mutations and/or stop codons may be produced in the mouse Brca2 gene, augmenting the effect of deletions within the mouse Brca2 gene.

Site-directed mutagenesis may be used to simultaneously construct a specific deletion and insert a linker sequence by using single stranded oligonucleotide to "loop-out" the desired region of the target gene (Krogstad and Champoux 1990, J. Virol. 64 (6):2796-2801, herein incorporated by reference).

The mutation engineered in the targeting vector can contain DNA sequences between the regions of Brca2 gene homology in addition to the positive selection marker, for example, splice acceptor sequences. Such sequences have been shown to facilitate aberrant splicing to create mutant message.

The DNA used as regions of homology should be derived from genomic DNA from the Brca2 gene locus from the mouse or sequences that flank the Brca2 gene locus. The strain of mouse from which the DNA derives is not important but preferably it should be the same as the strain of mouse from which the cells derived in which the gene targeting will be performed. Using DNA for the homology regions which is isogenic to the cells the cells in which the gene targeting will be performed may enhance the efficiency with which gene targeting is accomplished. The regions of homology may be derived from genomic libraries of mouse DNA which may be cloned into a variety of library vectors such as lambda phage vectors, cosmid vectors, plasmid vectors, pl phage vectors, 18 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 yeast artificial chromosome vectors, or other vectors.

Regions of homology to be used in the targeting vector could also be derived directly from genomic DNA using the polymerase chain reaction (PCR). This method relies on having some knowledge of the sequence of the Brca2 gene which is published (Sharan and Bradley 1997, Genomics 40:234-241).

Regions of homology so derived could be subcloned directly into the targeting vector.

The particular cloning vector employed in the present invention to construct the targeting vector comprising two regions of Brca2 homology separated by a positive selectable marker gene and an optional flanking negative selectable marker is not critical as long as the cloning vector contains a gene coding for a selective trait, e.g. drug resistance.

Examples of such cloning vectors include pBR322 and pBR322based vectors (Sekiguchi, 1983 Gene 21:267), pMB9, pBR325, pKH47 (Bethesda Research Laboratories), pBR328, pHC79, phage Charon 28 (Bethesda Research Laboratories, Boehringer Mannheim Biochemicals), pKB11, pKSV-10 (P-L Biochemicals), pMAR420 (Otsuka, 1981) and oligonucleotide (dg)-tailed pBR322 (Bethesda Research Laboratories), pBluescript or similar plasmids (Stratagene), pucl9_or similar plasmids (New England Biolabs).

The targeting vector comprising two regions of Brca2 homology separated by a positive selectable marker gene and an optional flanking negative selectable marker could be cloned into other cloning vectors such as lambda phage vectors, cosmid vectors, plasmid vectors, pl phage vectors, yeast artificial chromosome vectors, or other vectors.

Another option is to prepare the components of the targeting vector synthetically by PCR and simply ligating each component into its proper position by choosing restriction endonuclease sites for ligation which insured proper orientation of the homology regions relative to each other, and to insure that the positive selectable marker was located between the regions of homology.

19 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 Cloning vectors, other than the ones described in figure 1, containing unique cloning sites which are useful in the present invention can be determined upon evaluation of restriction nucleases. Other restriction nucleases which can be employed to produce fragments containing the mouse Brca2 gene, and thus other cloning vectors which can be useful in the present invention, are readily apparent from the mouse Brca2 gene restriction map. In fact, many combinations of restriction endonucleases could be used to generate an Brca2 targeting vector to mutate the Brca2 gene. These regions of homology could be cloned into any of a large number of commercially available plasmids such as the pBluescript series (Stratagene), the puc series (New England Biolabs), or the pGEM series (Promega).

The specific host employed for-growing the targeting vectors of the present invention is not critical. Examples of such hosts include E. coli K12 RR1 (Bolivar et al., 1977, Gene 2:95); E. coli K12 HB101 (ATCC No. 33694); E. coli MM21 (ATCC No. 336780); and E. coli DH1 (ATCC No. 33849). The preferred host in the present invention is DH5alpha (Life Technologies). Similarly, alternative vector/cloning systems could be employed such as targeting vectors which grow in E.

coli or Saccharomyces cerevisiae, or both, or plasmid vectors which grow in B. subtilus (Ure et al., 1983, Methods of Enzymology, "Recombinant DNA", vol. 101, Part C, Academic Press, N. The specific mouse cell which is mutated in the present invention is not critical thereto, and is preferably a precursor pluripotent cell. The term precursor means that the pluripotent cell is a precursor of the desired transfected pluripotent cell which is prepared in accordance with the present invention. The pluripotent cell may be cultured in vivo to form a mutant mouse (Evans et al., 1981, Nature 292:154-156). Examples of mouse cells which can be employed in the present invention include embryonic stem (ES) cells (preferably primary isolates of ES cells), such as AB1 or AB2.1. Primary isolates of ES cells may be obtained 20 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCTIUS98/17566 directly from embryos, such as described for the EK.CCE cell line or for ES cells in general. The particular embryonic stem cell employed in the present invention is not critical thereto. Examples of such embryonic stem cells are AB 2.1, an hprt- cell line, AB 1, an hprt' cell line. Other selectable markers such as those outlined in Table I could be used in other stem cell lines.

The ES cells are preferably cultured on stromal cells, STO cells and/or primary embryonic fibroblast cells as described by Robertson, 1987, In "Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp. 71-112. The stromal (and/or fibroblast) cells serve to reduce the clonal outgrowth of abnormal ES cells.

In order to obtain the Brca2-impaired mice of the present invention, the mutant embryonic stems cells are injected into mouse blastocysts as described by Bradley, 1987, In "Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp. 113-151.

The particular mouse blastocysts employed in the present invention is not critical thereto. Examples of such blastocysts include those derived from C57BL6 mice, C57BL6Albino, Swiss outbred, CFLP, MFI or others. Mice heterozygous for the brca2 mutant allele generated from the injected blastocyst can be screened for mutations in the Brca2 gene, by Southern blotting using DNA probes for said mutation (Figure or by PCR.

The mutant mice of the present invention can be intercrossed to obtain embryos homozygous for the mutation in the brca2 gene, and/or can be crossed with other mice strains to transfer the brca2 mutation into these other strains.

The following examples serve to more fully describe the manner of making and using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is Sunderstood that these examples in no way serve to limit the 21 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLES

Embryonic stem cells were manipulated essentially as described by published procedures (Teratocarcinomas and embryonic stem cells: a practical approach, E. J. Robertson, ed., IRL Press, Washington, D. 1987; Zjilstra et al., 1989, Nature 342:435-438; and Schartzberg et al., 1989, Science 246:799-803, each of which is herein incorporated by reference).

DNA cloning procedures were carried out essentially as described in J. Sambrook, et al. in Molecular Cloning: A Laboratory Manual, 2d ed., 1989, and periodic updates thereof, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N. which is incorporated herein by reference).

Oligonucleotides were synthesized on an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.

6.1. Cloning of the Mouse Brca2 Gene The mouse Brca2 gene was cloned from a mouse 129SvEv strain genomic library. More specifically, a fragment of the Brca2 gene was obtained using oligonucleotides based on sequence and reverse transcriptase polymerase chain reaction on RNA from mouse cells. The fragment of the mouse gene so obtained was subcloned into a plasmid vector pBluescript SK+ (Stratagene). A radiolabeled probe was made using that subclone of the Brca2 gene. The probe was used to screen a mouse 129SvEv-strain genomic lambda phage library to identify phage containing the homologous mouse gene. Three positive phage were isolated, grown, and restriction mapping performed on the DNA inserts by standard techniques.

22 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 6.2. Construction of Targeting Vectors To generate Brca2-impaired mice, two targeting vectors were constructed. pMB2TVhprt was used to generate the brca2l exl allele (Figure la): the vector contains 5.4 kb of DNA homologous 5' to exon 27 of the mouse Brca2 gene, and 1.9 kb of DNA homologous 3' to exon 27 of the mouse Brca2 gene.

This vector also contains a marker for positive selection (the Hypoxanthine phosphoribosyltransferase, HPRT, minigene cassette), and a marker for negative selection (the thymidine kinase, tk, gene).

More specifically, based on the restriction map generated, a region of homology upstream and downstream to exon 27 of the mouse Brca2 gene. The upstream homology region was isolated by an Apal and SacI digest (Figure la) which released approximately a 5.4 kb DNA fragment. The downstream homology region was isolated by an HindIII and Smal digest (Figure la) which released approximately a 1.9 kb DNA fragment.

To prepare pMB2TVhprt, a 2.5 kb genomic fragment from SacI to HindIII and containing coding nucleotides 9420-9984 were removed and replaced with the positive selectable marker. To prepare a positive-negative selection targeting vector, the negatively selectable tk gene was added exterior to the 3' homology region. The KpnI site which was unique and used to cut the vector prior to transfection (Figure la).

To generate the brca2l ex2 allele: the vector (pMB2TVneo) contains 4.6 kb of DNA homologous 5' to exon 26 of the mouse Brca2 gene, and 1.9 kb of DNA homologous 3' to exon 27 of the mouse Brca2 gene. This vector also contains a marker for positive selection (the neomycin phosphotransferase cassette), and a marker for negative selection (the tk gene).

More specifically, based on the restriction map generated, regions of homology upstream of exon 26 (including only a small fraction of the 5' part of exon 26) of the mouse Brca2 gene and downstream to exon 27 of the mouse Brca2 gene were used. The upstream homology region was isolated by an Apal and Clal digest (Figure Ib) which released a DNA 23 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 fragment of approximately 4.6 kb. The downstream homology region was isolated by an HindIII and Smal digest (Figure Ib) which released a DNA fragment of approximately 1.9 kb.

To prepare pMB2TVneo, a 3.3 kb genomic fragment from Clal to HindIII and containing coding nucleotides 9265-9984 was removed and replaced with the positive selectable marker.

To prepare a positive-negative selection targeting vector, the negatively selectable tk gene was added exterior to the 3' homology region. A unique KpnI site was used to cut the vector prior to transfection (Figure Ib).

6.3. Transfection of Mouse Embryonic Stem Cells Homologous recombination of the targeting vector with the Brca2 genomic locus was effected in mouse embryonic stem cells deficient for Hprt activity (See Figure More specifically, 10 Ag of the positive-negative targeting vector obtained in section 6.2 above was transfected into 1 x 107 129SvEv mouse strain embryonic stem cells deficient for Hprt activity and the resulting cells were grown in HAT (Hypoxanthine, Aminopterin, Thymidine) selection media to select for those cells which were transfected with the targeting construct to generate the brca2 ex l allele. Negative selection against the tk gene was also applied using the drug FIAU so as to enhance selection for those cells which had undergone a homologous recombination event at the Brca2 locus. Surviving colonies were screened by mini-Southern, as described by Ramirez-Solis, 1992, Anal. Biochem. 201:331-336, using a fragment of DNA from the Brca2 locus which was 3' to the region of homology of the targeting vector as probes so as to detect the double reciprocal homologous recombination event between the targeting vector and the Brca2 locus in the chromosome of the ES cell. Genomic DNA was digested with BglII and separated by electrophoresis. The desired recombination event was detected using the 3' probe which revealed a mutant allele of 2.9 kb for the pMB2TVhprt vector after gene targeting and a mutant allele of 9.4 kb for the pMB2TVneo vector after gene targeting as compared to the wild 24 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 type allele of 6.0 kb. Many positive ES cell clones were identified as correct replacement events, with an approximate kb genomic deletion after gene targeting with the pMB2TVhprt vector and a 3.3 kb genomic deletion after gene targeting with pMB2TVneo.

Clones of targeted ES cells with the Brca2 exl allele were subsequently targeted with the vector to generated the Brca2 1ex2 allele. After transfection with 10 gg of vector the cells were selected in G418 selection media to select for those cells which were transfected with the targeting construct to generate the brca2 1e 2 allele. Negative selection against the tk gene was also applied using the drug FIAU so as to enhance selection for those cells which had undergone a homologous recombination event at the Brca2 locus. Surviving colonies were screened by mini-Southern, as described by Ramirez-Solis, 1992, Anal. Biochem. 201:331-336, using a fragment of DNA from the Brca2 locus which was 3' to the region of homology of the targeting vector as probes so as to detect the double reciprocal homologous recombination event between the targeting vector and the Brca2 locus in the chromosome of the ES cell. ES cell genomic DNA for the minisouthern was digested with restriction enzyme BglII (Figure la,b). In addition, many of these targeted clones were mutated at both Brca2 alleles to generate brca2exl/brca2 ex2 compound heterozygotes.

6.4 Generation of Brca2 Impaired Mice and Embryonic Fibroblasts ES cell clones representing the following genotypes, brca2ex/+, brca2lex2/+, brca2ex/brca2 1 x2 as obtained in section 6.3 above were injected into C57BL6 Albino host blastocysts as has been described by Bradley, 1987, In "Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp. 113-151.

Injected blastocysts were implanted into pseudopregnant females and chimeric offspring were born as demonstrated by the mixture of agouti and albino coat colors (agouti 25 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 contribution from the ES cell line and albino from the wildtype host embryos). Chimeric male mice were mated to wildtype C57BL6 Albino females and agouti pups were born indicating successful germline transmission of the ES cell component of the chimeric mouse, resulting in C57BL6 Albino/129SvEv hybrids (referred to as C57BL6/129 hybrids).

At three weeks of age, the offspring from the chimeric crosses were screened for the mutant brca2 alleles as described below.

Genomic DNA was isolated from the resulting mice. Then (g of the resulting genomic DNA was digested with BglII, and subjected to Southern blot analysis using the 3' probe as described above for the minisoutherns. ES cell clones transmitted the mutant allele through the germline for both brca2l exl and brca2 1 ex2 alleles. A male and female mouse were identified heterozygous for the mutant allele.

The male and female mice found to be heterozygous for the brca2 mutations were intercrossed. The chimeric mice were also bred to 129SvEv strain mice, in order to place the mutant allele on the 129SvEv strain background.

The brca2~"' mutation is most likely not null The messenger RNA was analyzed to determine whether the brca2 ex2 allele produced an altered transcript. RT-PCR (reverse transcriptase-polymerase chain reaction) was employed with one primer in exon 26 of mouse Brca2 gene and another in exon 3 of the HPRT minigene. A fusion transcript was detected such that the sequences found in exon 27 of mouse Brca2 were deleted and exon 26 sequences of mouse brca2 were fused to exon 3 of the HPRT minigene. An additional amino acid is coded by the HPRT minigene sequences before a stop codon terminates translation (Figure Ic).

26 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 6.6. brca21"2/brca2'2 Compound Heterozygous ES Cells are Hypersensitive to Ionizing Radiation but not UV light The brca2 ex I/brca2 1e 2 compound heterozygous cells were tested for their ability to repair damage caused by two genotoxic agents, y-radiation and UV light (Figure For the y-radiation analysis (Figure 2a): controls were wildtype Hprt positive cells (one clone), wild-type Hprt deficient cells (three clones), brca2 e l cells (eight clones), brca21 x 2 cells (six clones). No difference was found between these clones so their numbers were averaged.

Eight clones of brca2' e l /brca2z 1 2 cells were observed and averaged. At 250 RADS, 500 RADS and 750 RADS there were and 10 fold fewer brca21exl/brca21ex 2 colonies to survive as compared to controls. For the UV light analysis (Figure 2b): controls were brca2ex l cells (three clones) and brca21ex 2 cells (two clones). No difference was found between these clones so their numbers were averaged. Five clones of brca2exl/brca2 1 ex 2 cells were observed and averaged. Both control and brca2 1 exI/brca2 ex2 cells exhibited the same degree of sensitivity to UV light. Therefore, the brca2 1 e xl /brca2 1 e 2 genotype exhibits increased sensitivity to an agent that induces breaks in DNA (y-radiation) but not to an agent that induces pyrimidine dimers (UV light).

6.7. brca21~I/brca2' 2 Compound Heterozygous Embryonic Fibroblast Cells Undergo Premature Replicative Senescence Mouse embryonic fibroblasts (MEF) were analyzed for proliferation and life span (figure Control MEF were derived from 129SvEv embryos. brca2 ex /brca21ex2 MEF were derived from chimeric 15.5 day embryos (Swiss Webster recipient embryos injected with brca2 1 xl brca2 1ex2 129SvEv cells). Chimerism was identified by embryos with black eyes since Swiss Webster is albino. brca21exl/brca21ex2 MEF were from chimeric embryos by growing in G418 for ten days which selected for expression of the neo cassette. The proliferation characteristics was determined for control and 27 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 brca2exl/brca2ex 2 MEF. To ensure the brca21exl/brca2 1ex2

MEF

were not contaminated with cells derived from the Swiss embryo, they were grown with and without G418 (there was no contamination because no difference in proliferation was observed).

A growth curve was established for control and brca21exl/brca2 1 ex 2 MEF plated at high density (8 X 104 cells/ cm plate). The control MEF grew slightly faster than brca21exl/brca21x2 MEF at high density (Figure 3a) indicating that the brca2 1 exI/brca2z 1e 2 suffered from a growth disadvantage. MEF were labeled with BrdU and stained with propidium iodide to measure the number of cells that go into S phase over a period of 48 hours. About 10 20% fewer brca2 1 exl/brca21e x2 MEF entered S phase than control MEF, indicating that a higher percentage of brca21exl/brca21ex2 MEF were senescent (Figure 3b).

Proliferative ability was established for control and brca2exl/brca2ex 2 MEF plated at low density. The difference in proliferation was much more dramatic at low density (Figure 3c). The total number of colonies (includes all colonies with >3 cells) was counted and there were 10 fold fewer brca21 e l/brca21ex 2 colonies than control colonies when 5000 MEF were plated onto a 10 cm plate. A percentage of these colonies, ones with >15 cells, was determined for a colony size distribution (CSD). The CSD is an accurate indication of the cell's life span. Again there was a fold decrease in the CSD for brca2 exl /brca21ex 2 MEF compared to control MEF (Figure 3d).

Life span of the control and brca2lexl/brca2 ex2 MEF was established (Figure 3e). The CSD indicates that the brca2 1 ex1/brca2ex 2 MEF will undergo senescence faster than the control MEF. MEF were plated (1 X 105 cell/ 3.5 cm plate) onto three plates. They were passaged every 3.5 days and replated at the same concentration. As the number of cells decreased, then the same number of cells was plated onto fewer plates until there were no longer enough cells to plate onto a single plate. At this point the cells are considered 28 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 senescent. The brca2 1 ex l /brca2 ex2 MEF were shown to become senescent at passage 7 8 while the control MEF could be passaged longer. In addition, one control MEF spontaneously immortalized. Thus the brca2exl/brca21e 2 MEF undergo premature replicative senescence.

6.8. Screen for rescue of proliferation/senescence defect in Brca2 impaired cells Embryonic fibroblasts impaired for Brca2 function will be used to screen for genetic mutations that rescue the proliferation/premature replicative senescence defect. The mutations may be made by a variety of techniques and the particular technique employed to make the mutations is not important. Examples of methods to make mutations is to expose the cells to DNA damaging agents, preferably agents that do not generated double-strand breaks because it is likely that double-strand breaks will be lethal to these cells. Another method is to infect with retrovirus. The integration of the retrovirus will introduce mutations.

Another approach to rescue the proliferation is to ectopically express transgenes in the fibroblasts impaired for Brca2 function. A variety of expression libraries may be used and the particular kind of library is unimportant.

Another approach to rescue the poor proliferation/ premature replicative senescence phenotype is to induce overexpression of endogenous genes in the fibroblasts impaired for Brca2 function. A variety of techniques may be used and the particular kind of technique is unimportant.

6.9. Mice and Cells Impaired for Brca2 Function as Animal Models for Cancer and to Test the Mutagenicity of Toxic Agents Mice and cells that are impaired for Brca2 function may be used as a model system for oncogenesis and to test the mutagenicity of genotoxic agents. Brca2 impaired mice may be observe for onset and type of cancer. Brca2 impaired mice may be bred to mice with known predispositions to cancer, such as p53-mutant mice to observe a change in the onset of 29 SUBSTITUTE SHEET (RULE 26) WO 99/10479 PCT/US98/17566 cancer or the spectrum of cancer. Brca2-impaired mice and cells may be exposed to a variety of toxic gents to test for mutagenicity by onset and spectrum of tumor formation or by observing cell viability, proliferation and chromosomal damage.

All publications, patents, and patent applications mentioned in the above specification and herein incorporated by reference. Various modifications and variations of the described invention will be apparent to those skilled of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described methods, techniques, and cells and animals are intended to be within the scope of the following claims.

30 SUBSTITUTE SHEET (RULE 26)

Claims (19)

1. A non-human diploid animal cell containing an engineered mutation in at least one allele of the Brca2 gene located in a region of the Brca2 gene downstream from nucleic acid residue 9265 of Genbank Accession No. U65594 and wherein the mutation produces a protein that is impaired for its ability to directly or indirectly associate with MmRad51.

2. A cell according to claim 1, wherein the cell is homozygous for the 10 mutant Brca2 allele.

3. A cell according to claim 1 or 2, wherein said mutation is a deletion mutation.

4. A non-human diploid animal cell containing a first engineered mutation in one allele of the Brca2 gene and a second mutation engineered in the other allele of the Brca2 gene wherein the mutations produce a protein 09 o Sthat is impaired for its ability to directly or indirectly associate with 2 2MmRad51. •o

5. A non-human transgenic animal which comprises an engineered mutation which decreases the expression or alters the function of at least one allele of the Brca2 gene, wherein said mutation is located in a region of the Brca2 gene downstream from nucleic acid residue 9265 of GenBank Accession No. U65594 and wherein the mutation produces a protein that is impaired for its ability to directly or indirectly associate with MmRad51.

6. A mutant embryo offspring of the non-human transgenic animal according to claim -32-

7. A transgenic animal according to claim 5 that is homozygous for said mutation in the Brca2 gene.

8. A transgenic animal according to claim 5 or 7, wherein said mutation is a deletion mutation.

9. A method for identifying a gene, which in a mutant form rescues the premature replicative senescent phenotype of cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, comprising the steps of: introducing at least one genetic mutation into the cells; measuring proliferation ability of the mutated cells using 3T3, 3T9, or population doubling analysis; isolating the mutated cells that are not senescent after passage 8; and identifying the gene in the cells which has been mutated in step

10. A method to rescue cells containing an engineered mutation in at least one allele of the Brca2 gene from premature replicative senescence, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MnRad51, comprising underexpressing an endogenous gene identified in claim 9.

11. A method according to claim 10, wherein the cells containing an engineered mutation in at least one allele of the Brca2 gene have a genetic condition selected from deficiency in negative regulation of the cell cycle, proficiency in negative regulation of the cell cycle, proficiency in progression through the cell cycle, or decreased life-span of a cell or S organism. No DeletelAmendments\RN609573-S.12.02.doc 5 December 2002 -33-

12. A method for identifying a compound that rescues cells containing an engineered mutation in at least one allele of the Brca2 gene from premature replicative senescence, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, comprising the steps of: contacting the cells with a compound; and measuring the proliferation ability of the cells using 3T3, 3T9, or population doubling analysis; whereby the presence of mutated cells that are not senescent after passage 8 identifies the compound.

13. A method to increase the incidence of cancer in an animal having cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, comprising: underexpression of the gene identified in claim 9, in the animal.

14. A method to decrease the incidence of cancer in animals having cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, said method comprising one or more steps selected from: overexpression of a gene identified in claim 9 being transfected into the cells of the animal; overexpression of an endogenous gene identified in claim 9; ectopic expression of a gene identified in claim 9 being transfected into the cells of the animal; or ectopic expression of an endogenous gene identified in claim 9. A method for screening for compounds or molecules that increase the R incidence of cancer in animals having cells containing an engineered i Not Deletev\AmndmentsJRN609573-5.12.02.doc 5 December 2002 -34- mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad51, comprising the step of exposing the animals to said compounds and identifying a compound or molecule that increases the incidence of cancer in the animals.

16. A method of screening for compounds or molecules that decrease the incidence of cancer in animals having cells containing an engineered mutation in at least one allele of the Brca2 gene, wherein the mutation results in a Brca2 protein that is impaired for its ability to directly or indirectly associate with MmRad5l, comprising the step of exposing the animals to said compounds and identifying a compound or molecule that decreases the incidence of cancer in the animals.

17. A diploid animal cell according to claim 1 substantially as hereinbefore described with reference to Example 6.3.

18. A diploid animal cell according to claim 4 substantially as hereinbefore described with reference to Example 6.3.

19. A transgenic animal according to claim 5 substantially as hereinbefore described with reference to Example 6.4. A method according to claim 9 substantially as hereinbefore described with reference to Example 6.7 or 6.8.

21. A method according to Claim 12 substantially as hereinbefore described with reference to Example 6.8. DATED: 6 December 2002 PHILLIPS ORMONDE FITZPATRICK Neys for: LEX[L N GENETICS INCORPORATED