We will perform insertional mutagenesis using murine stem cell virus (MSCV) to activate genes in ES cells using the viral LTR. The advantage of using retroviruses for insertional mutagenesis is that cloning of the insertion sites is relatively simple. ES cells with randomly inserted MSCV will be selected for viable clones after both copies of the functional Brca1 or Brca2 are disrupted. The advantage of using MSCV is that, unlike other retroviral vectors, its LTR can drive high levels of target gene expression in ES cells. In a complementary approach, we will also use an shRNA library-based screen to identify genes whose loss of function can rescue the BRCA1- or BRCA2-deficient ES cells. We will also use this genetic screen to rescue the lethality of ES cells expressing deleterious variants that disrupt a specific biological process, e.g. a variant known to be defective in the E3 ubiquitin ligase activity of BRCA1. It will be interesting to compare the suppressors identified in such cells with those identified in ES cells that completely lack BRCA1. We have identified a number of candidate genes that can rescue lethality of Brca2 null ES cells. Our current focus is on understaning the mechanism and the physiological relevance of these genetic interactors.
