Citation

Germline mutations in the BRCA1 gene are one of the major causes of familial and ovarian cancers.
Although the organ-specific carcinogenic mechanism has not been elucidated, it is thought that the estrogen
receptor α (ERα) plays a role based on the histological findings of the origin of cancer.
Therefore, in order to elucidate the relationship between the two factors in carcinogenesis,
it is important to analyze the effect of ERα on BRCA1 dysfunction using normal diploid cells.
However, there are no strained ERα-positive diploid cells. Therefore, in this study, we established
ERα-positive BRCA1-depleted human mammary gland cells using genetic engineering techniques as a model
cell line suitable for analysis. Dox-ERα cells were established. Furthermore, after infecting MCF10A-Dox-ERα
cells with CS-RfA-ETBsd-shBRCA1-derived wrench virus, MCF10A-Dox-ERα-shBRCA1 cells were established under the
selection of both Blasticidin and Puromycin antibiotics. It was confirmed that the addition of Dox reduced the
expression of BRCA1 and at the same time expressed ERα in MCF10A-Dox-ERα-sh BRCA1 cells.
Finally, the effect of ERα on BRCA1 depletion was examined using cell proliferation ability as an index.
Interestingly, ERα expression did not affect cell proliferation in BRCA1-normally expressing cells,
whereas ERα expression significantly reduced cell proliferation in BRCA1-depleted cells.
As described above, Dox-inducible ERα-positive BRCA1-depleted human mammary gland cells were established.
In the future, it will be a useful tool for analyzing the effect of ERα on gene instability against the background of BRCA1 dysfunction.
First, doxycyclin (Dox) -induced BRCA1-depleted cells were generated using MCF10A, an ERα-negative normal
mammary gland cell line. The shRNA sequence for BRCA1 was cloned into an entry vector, and the CS-RfA-ETBsd-sh
BRCA1 wrench viral vector was prepared by the Gateway method. After infecting MCF10A cells with a wrench virus
prepared from Lenti-X 293T cells, MCF10A-Dox-sh BRCA1 cell line was established by selecting with Blasticidin.
Similarly, MCF10A-, which produces CSIV-TRE-Rfa-Ubc-puro-ERα-derived wrench virus and expresses ERα in a
Dox-inducible manner under the selection of Puromycin.