Epitope tagging of endogenous BRCA2 utilizing AAV and TALEN based constructs

Rsrch Assoc Ther Radiology
15 York St, New Haven, CT 06510-3221

The proper regulation of DNA double-strand break (DSB) repair is essential to the genome of every living organism. Deregulated DSB repair can lead to widespread genomic instability and eventual tumorigenesis.  BRCA2 has a firmly established role in DNA DSB repair and functional failures in this protein lead to cancer of the breast, ovaries and certain epithelial tissues. The lack of a genetically defined human cell model for BRCA2 function has hampered progress in understanding how loss of this gene drives tumor progression.  One of our goals is to create an isogenic human mammary epithelial cell system wherein the endogenous BRCA2 alleles can be modified and expression levels controlled.  To accomplish this, we have combined adeno-associated viral (AAV) gene targeting vectors with TALE-conjugated nuclease pairs (TALENs) to directly modify the endogenous alleles of BRCA2. One of our strategies will employ epitope tagging the destabilization domain (DD) from the ProteoTuner system to the N-terminus of BRCA2. This degron-regulated cellular switch will facilitate studies in both the “BRCA2 ON” (+ Shield1) and “BRCA2 OFF” (- Shield1) states.  In addition, we are epitope tagging the N-terminus of the endogenous BRCA2 alleles with the 2XMBP affinity tag to facilitate the detection of novel proteins that interact with BRCA2 after DNA damage.