Abstract
Germline mutations of BRCA1 cause familial breast and ovarian cancer. The mechanism responsible for the tissue specificity is unknown, although it is considered that estrogen receptor α (ERα) plays a critical role. However, lack of a diploid cell line expressing ERα has prevented researchers from analyzing the impact of ERα on generating genomic instability in the cells with BRCA1 deficiency. To overcome this problem, here we established BRCA1 deficient and ERα positive human breast cell line using biogenetic technology. First we generated doxycyclin (Dox)-inducible BRCA1-defective cells from MCF10A, an ERα-negative normal breast cell line. CS-RfA-ETBsd-shBRCA1 lentiviral vector was generated from oligonucleotides responsible for shRNA sequence in BRCA1 by Gateway recombination from pENTR4-H1tetOx1 entry vector. MCF10A-shBRCA1 cells were established from CS-RfA-ETBsd-shBRCA1-lentivitral infected MCF10A cells with blasticidin selection. Next we generated Dox-inducible ERα-expressing cell line from MCF10A cells by infection of CSIV-TRE-Rfa-Ubc-puro-ERα lentivirus followed by puromycin selection (MCF10A-ERα cells). Finally MCF10A-ERα-shBRCA1 cell line was established by infection of MCF10A-ERα cells with CS-RfA-ETBsd-shBRCA1 followed by double selection with blasticidin and puromycin. The effective inhibition of BRCA1 expression and simultaneous expression of ERα was verified by immunoblotting. Interestingly, whereas ERα expression had no effect on the proliferation of BRCA1 expressing cells, it dramatically suppressed the proliferation of BRCA1-defective cells. Thus, we established a human breast cell line with conditional inhibition of BRCA1 with simultaneous expression of ERα by Dox. This material would be valuable for analyzing ERα-induced genomic instability specifically occurring in a BRCA1-deficient genetic background.
