Identifying the effects of BRCA1 mutations on homologous recombination using cells that express endogenous wild-type BRCA1.

ABSTRACT: The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This deplet

SUBMITTER: Parvin J 

PROVIDER: S-EPMC3197403 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature