To evaluate the functional significance of human BRCA1 and BRCA2 variants and to carry out functional dissection of these proteins, we have developed a simple, reliable and physiologically relevant functional assay using mouse embryonic stem (ES) cells. The assay is based on the fact that Brca1 and Brca2-null ES cells cannot survive. Therefore we engineered ES cells to express a conditional allele of Brca1 or Brca2. Desired mutations are generated in the human BRCA1 or BRCA2 gene in bacterial artificial chromosomes (BAC) and introduced into the ES cells. Functional analysis is performed after deletion of the conditional allele. The ability of and the extent to which specific variants complement the lethality of ES cells associated with Brca1 or Brca2 deficiency is used to evaluate their functional significance. Variants that can rescue lethality are then screened for a defect in the known functions of BRCA1 and BRCA2.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Chang, Suhwan; Sharan, Shyam K (2013) The role of epigenetic transcriptional regulation in BRCA1-mediated tumor suppression. Transcription 4:24-8
Biswas, Kajal; Stauffer, Stacey; Sharan, Shyam K (2012) Using recombineering to generate point mutations:galK-based positive-negative selection method. Methods Mol Biol 852:121-31
Chang, Suhwan; Stauffer, Stacey; Sharan, Shyam K (2012) Using recombineering to generate point mutations: the oligonucleotide-based "hit and fix" method. Methods Mol Biol 852:111-20
Biswas, Kajal; Das, Ranabir; Eggington, Julie M et al. (2012) Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 21:3993-4006
Chang, Suhwan; Sharan, Shyam K (2012) Epigenetic control of an oncogenic microRNA, miR-155, by BRCA1. Oncotarget 3:5-6
Chang, Suhwan; Sharan, Shyam K (2012) BRCA1 and microRNAs: emerging networks and potential therapeutic targets. Mol Cells 34:425-32
Biswas, Kajal; Das, Ranabir; Alter, Blanche P et al. (2011) A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell-based assay. Blood 118:2430-42
Kuznetsov, Sergey G; Liu, Pentao; Sharan, Shyam K (2008) Mouse embryonic stem cell-based functional assay to evaluate mutations in BRCA2. Nat Med 14:875-81