Functional Analysis of Variants of Unknown Significance in BRCA1 and BRCA2 Using Complementation of a Synthetic Lethal Interaction with PARP Inhibition

: We continued to test MCF10A cells containing an inducible shRNA directed against the 3-UTR of BRCA2 for synthetic lethality with PARP inhibitors. Antibody staining with RAD51 and H2AX was used to look for double strand breaks and stalled replication forks and showed increase in RAD51 and H2AX positivity with knock-down of BRCA2 and PARP inhibitors. We also tried very hard to increase transfection efficiency in order to test wild-type and mutant forms of BRCA2 but were not able to transfect the large BRCA2 construct into these cells. We tried lipofection of various types, electroporation, and magnetofection. None of these methods were successful