Hereditary mutations of the BRCA1 and BRCA2 genes confer a profound predisposition to breast and ovarian cancer in women. Since the identification of these genes by positional cloning, we have used gene targeting to develop experimental mouse models for Brca defects. Low-level gamma radiation studies (0.3 Gy) with the first generation of these mice have been completed and pathology analyses are in progress with several inbred strains, hemizygous Brca1-deficient, Brca2-deficient, Brca1-deficient x Brca2-deficient F1, and Brca2-deficient x mutant P53 F1 mice. These experiments suggest that haploinsufficiency for Brca mutations of exon 10 and/or 11 do not strongly predispose mice to mammary tumor development relative to their wild type littermates. Dietary exposures to diethylstilbestrol, a model environmental estrogen, also did not induce mammary tumors in Brca-deficient mice after six months (short-term carcinogen screening bioassay). Likewise, the mammary tumor response in Brca2-deficient mice was not enhanced in a rapid ENU protocol with Apc-deficient mice. In contrast, examination of mammary gland whole mounts from these Brca-deficient mice at various ages suggests an inhibition of ductal morphogenesis as compared to their respective normal littermates that mimics preliminary findings with human patients. Although the mammary ducts appear to elongate normally, lateral and side branching and alveolar bud formation are reduced in haploinsufficient Brca mice relative to wild type littermates. Development of Brca-deficient mouse models has been inhibited by the early embryonic lethality of most homozygous mutant mice. We have used the Cre-loxP system to specifically disrupt Brca2 in mouse mammary tissue by generating mice that carry loxP sites flanking Brca2 exon 27. This carboxy terminal domain of the Brca2 protein interacts directly with the Rad51 protein and is thought to be critical for maintaining genomic stability in response to DNA damage. Floxed Brca2 mice have been intercrossed with two lines of transgenic mice that express the Cre recombinase under the control of the Mouse Mammary Tumor Virus (MMTV) promoter. We have defined the tissue specificity of Cre-mediated deletions at various timepoints through crosses between the MMTV-Cre strains and ROSA26LacZ-Cre reporter mice. Female pups from these MMTV-Cre crosses delete both floxed Brca2 exon 27 alleles homozygously in mammary epithelial cells during puberty. Offspring from these crosses have been followed ~1 year for spontaneous neoplastic development. In addition, a long-term radiation study with these Brca2-floxed mice has been initiated. We have also deleted exon 27 of Brca2 in floxed embryonic stem cells by transient Cre expression and generated Brca2 mice null for exon 27. Intercrosses of these mice with BALB/c P53-deficient mice are also being irradiated and followed for neoplastic development in mammary gland and other tissues. Finally, transfer of this Brca2 exon 27 mutation onto susceptible backgrounds (BALB/c and SWR) by speed congenic techniques is in progress to ask whether genetic modifiers may act synergistically to facilitate mammary tumorigenesis in Brca2-deficient mice. Mice that are homozygous for floxed or deleted Brca2 exon 27 alleles should provide an invaluable source of primary cells and cell lines for biochemical studies of Brca2 function in vitro.
