逆转录病毒感染结合流式细胞分选方法构建稳定过表达CUEDC2的MCF-10A细胞系

摘要

CUEDC2 (CUE domain containing protein 2) is a functionally unknown protein. In previous work, CUEDC2 interacts with the progesterone receptor (PR) and promotes progesterone-induced proteasomal degradation of PR. Further study suggested that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer and over-expressed in many tumors. In order to address the question if CUEDC2 affect the proliferation or migration of breast cancer cells, a stable cell line is constructed with the normal glandular epithelium cell, MCF-10A, by retrovirus. Cells with GFP expression indicating the expression of CUEDC2, thus, the GFP positive cells were selected by fluorescence-activated cell sorting. By combining the retrovirus infection and cell sorting, the selecting efficiency was markedly increased. This cell line will largely benefit the study of CUEDC2 in breast cancer.%CUEDC2( CUE domain containing protein 2)是本实验室发现的功能未知蛋白质.已有研究证明CUEDC2通过抑制孕激素受体PR影响乳腺癌细胞的生长,同时CUEDC2的高表达能够降解雌激素受体ER而导致乳腺癌内分泌治疗耐药.为了深入研究CUEDC2在乳腺癌中的功能,构建了稳定过表达CUEDC2的MCF-10A细胞系.利用逆转录病毒将非融合形式的绿色荧光蛋白质GFP和CUEDC2基因稳定整合至靶细胞的基因组中,经过流式分选获得GFP阳性细胞,即为稳定表达CUEDC2的细胞株.上述方法高效快捷,极大提高了稳定细胞株的构建效率.同时该稳定细胞株的建立为CUEDC2的功能研究提供了必要的工具.