Inherited defects of the BRCA1 and BRCA2 genes confer a profound predisposition to breast and ovarian cancer in women. Since the identification of these breast cancer susceptibility genes by positional cloning, we have used gene targeting to develop experimental mouse models for defects in the Brca2 gene. Our studies have focused on mice lacking the carboxy terminal domain of the Brca2 protein (exon 27) because this portion of the gene product has been shown to contain nuclear localization signals and it interacts directly with the Rad51 protein which is thought to be critical for maintaining genomic stability in response to DNA damage. Using homologous recombination techniques, we have generated mice that carry one (hemizygous ) or two (homozygous) mutant alleles of Brca2 exon 27. In contrast to previous attempts by ours and other laboratories to generate homozygous mutant Brca2 mice, our exon 27 null mice are viable although we have observed a subtle but statistically significant deficiency in the expected number of homozygous Brca2 mutant mice. An initial study with these mice has shown that homozygous mutant Brca2 mice on a C57BL/6J background are predisposed to a low incidence of spontaneous lymphoma development compared to their heterozygous and wild type littermates. High- and low-level gamma radiation studies (5 & 0.3 Gy at 5 wks of age) with intercrosses between these C57BL/6 Brca2-exon 27 deficient mice and BALB/c-P53-deficient mice are being followed for neoplastic development in mammary gland and other tissues. Preliminary results from this study indicate that tumor latency is decreased by 4 to 8 weeks in mice carrying the homozygous Brca2 exon 27 mutation relative to their littermates. While lymphomas dominate the tumor spectrum of these irradiated mice, at least one third of animals heterozygous for the Brca2 and P53 mutations develop mammary tumors by approximately 8 and one half months of age. In parallel studies, we have utilized the Cre-loxP system to specifically disrupt Brca2 in mouse mammary tissue by generating mice that carry loxP sites flanking Brca2 exon 27. Floxed Brca2 mice have been intercrossed with transgenic mice that express the Cre recombinase under the control of the Mouse Mammary Tumor Virus (MMTV) promoter. Studies with ROSA26-Cre reporter animals suggest that our intercross mice delete both floxed Brca2 exon 27 alleles homozygously in the vast majority of mammary epithelial cells during puberty. Surprisingly, neoplastic development in female offspring from this cross does not appear to be dramatically enhanced in the Cre+ versus Cre- mice after >1 year with or without 5 Gy irradiation. Examination of mammary gland whole mounts from virgin female mice with germline and conditional Brca2 mutations at various ages suggest an inhibition of ductal morphogenesis as compared to their respective normal littermates. This observation is intriguing because it mimics preliminary reports with women carrying BRCA mutations. Although the mammary ducts appear to elongate normally in our Brca2-deficient mice, lateral and side branching and alveolar bud formation are reduced relative to their wild type littermates. Finally, we are currently using fluorescent microsatellite marker-assisted breeding techniques (speed congenics) to transfer this Brca2 exon 27 mutation onto genetic backgrounds (BALB/c and SWR) that we have previously shown are highly susceptible to radiation-induced mammary carcinogenesis. Future studies by our group will attempt to determine whether genetic modifiers in the BALB/c and SWR mouse genomes may act independently or synergistically to facilitate mammary tumorigenesis in Brca2-deficient mice after irradiation.
