Checkpoint Functions of the BRCA1/BARD1 Tumor Suppressor

The objective of my research is to study the role of BRCA1 in breast cancer by determining how BARD1 phosphorylation affects the checkpoint and DNA repair functions of the BRCA1BARD1 heterodimer. In my original application, I proposed to examine the role of BARD1 phosphorylation in the checkpoint functions of BRCA1 by generating and characterizing isogenic subclones of HCT116 cells that express different knock-in alleles of BARD1. Subsequently however, I also tested the feasibility of an alternative approach based on siRNA-mediated depletion of endogenous BARD1 coupled to transient reconstitution with exogenous BARD1. This approach has several advantages over the original knock-in strategy. First, since it involves transient transfection of a cell population this approach is not susceptible to artifacts that arise due to clonal variation. Second unlike the knock-in strategy which is restricted to certain pseudo-diploid cell lines such as HCT116, this approach can be applied to a broad range of cell types. Third, this approach is more facile since it does not require the laborious process of generating stable knock-in subclones by targeted gene recombination.