Breast cancer is the most common disease and leading cause of lethality in women. The identification and cloning of the BRCA1 and BRCA2 genes have demonstrated that mutations in these two genes account for almost all of the families with multiple cases of breast and ovarian cancer. To understand the biological function of these genes, mutations that result in loss of function have been generated in mice. These studies have demonstrated the involvement of these two genes in cell proliferation and normal development and also in the Rad51-mediated DNA recombination repair machinery. Mutation in the Brca1 or Brca2 genes may result in a defect in the DNA repair/monitor system causing accumulation of mutation in growth control genes, leading to tumorigenesis. One of the goals of our research is to understand the function of various small conserved domains of Brca2 protein. To achieve this, we are generating subtle mutations in regions of the gene encoding these domains to study their phenotypic effect. To generate multiple alleles of the Brca2 gene, we are using bacterial artificial chromosomes (BACs). We have identified a BAC clone that contains the functional Brca2 gene, as it can rescue the embryonic lethality found in Brca2-deficient mice. We are using BACs with a mutation in the Brca2 gene to generate transgenic mice on a Brca2-deficient background. We are manipulating the BACs by using the bacteriophage recombination system. We have developed a rapid method to manipulate BACs to generate deletions, insertions, and single base changes using oligonucleotides as targeting vectors, thus obviating the use of selection markers. We have developed a PCR-based approach to identify the correctly targeted clone. In human cancer patients, in addition to mutations that clearly disrupt the gene, several missense mutations have been identified in the BRCA1 and BRCA2 genes. The functional significance of these mutations is unknown. These may result in an unstable protein, alter a subset of its function, or represent a rare polymorphic variant.
Our aim i s to generate missense mutations in murine Brca1 and Brca2 genes and study their functional significance using the BAC transgenic approach. We have examined the possibility of using the human gene in mice to generate missense mutations. We have generated humanized mice using human BRCA1 gene. These transgenic mice have a functional BRCA1 gene, which is able to rescue the embryonic lethality associated with mutation in the endogenous Brca1 gene. In addition to the murine gene, we are generating missense mutations in the human gene to examine their functional significance. Such mutations will serve as valuable tools for functional dissection of these genes. Interaction between Brca2 and Rad51 proteins was identified in a yeast two-hybrid screen, which proved to be valuable in understanding the role of Brca2 as a tumor suppressor. We are now using the yeast two-hybrid system to identify proteins that interact with various conserved domains of the Brca2 protein. This will help us in understanding their functional significance.
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