稳定表达TMEM16A蛋白TE-1细胞株的构建与鉴定

Transfection of TMEM16Agene into TE-1 cells stably by liposome and its identification

目的建立稳定表达TMEM16A蛋白的TE-1肿瘤细胞株。方法用Lipofectamine 2000转染试剂将TMEM16A基因转染到TE-1细胞,经G418筛选,使用激光共聚焦显微镜和Western blot技术检测TMEM16A基因是否在TE-1细胞中已经稳定表达。结果通过使用G418进行筛选,阳性克隆于4周后培养形成。激光共聚焦显微镜观察稳定表达TMEM16A蛋白的TE-1细胞可见细胞发出绿色荧光。Western blot结果表明,未稳转细胞和稳转细胞系统均表达有TMEM16A蛋白,而稳转细胞系统的蛋白水平明显高于未转染细胞系统的蛋白水平。结论利用脂质体转染方法成功构建了稳定表达TMEM16A基因的TE-1细胞系。

Objective To get stably expressing TMEM16A proteins by transfecting TE-1 cells. Methods TMEM16A gene was transfected into TE-1 cells by LipofectamineTM 2000, then we picked up cell clones in the presence of G418. TMEM16A proteins were identified by confocal microscope and western blot. Results After 4 weeks, the cell clones of colony-like formed after G418 screening. The stable TMEM16A-expression TE-1 cell lines were observed by confocal microscope showing signal of GFP co-transfected. Western blot results showed that TMEM16A protein expressed in control and stable TMEM16A-expression TE-1 cells, and the expression was significantly increased in TMEM16A stable cell lines. Conclusion Stable TMEM16A- expression TE-1 cell lines are created with liposome method successfully.