短发夹RNA载体稳定抑制端粒相关蛋白TERF2IP1表达的HCT116细胞的构建

Construction of Recombinant HCT116 Cells with sh RNA Vector Targeting TERF2IP1 Gene

目的:构建针对TERF2IP1基因的短发夹RNA(sh RNA)表达载体,在HCT116细胞中鉴定该载体对TERF2IP1基因的干扰效果。方法:设计针对TERF2IP1基因的sh RNA序列,将其克隆至p GPHI/Hygro载体中,瞬时转染HCT116细胞48 h后,用400 mg/m L的潮霉素筛选抗性克隆,获得阳性单克隆细胞株;分别提取细胞总RNA和蛋白,进行RT-PCR和Western印迹,检测TERF2IP1的表达水平。结果:构建了4对针对TERF2IP1基因的sh RNA表达载体,通过潮霉素筛选获得稳定干扰TERF2IP1基因的HCT116细胞系,实时定量PCR和Western印迹实验均证实潮霉素筛选后,HCT116细胞中TERF2IP1的表达水平明显降低。结论:构建了4对人TERF2IP1基因的sh RNA表达载体,获得4株分别针对TERF2IP1基因不同位点的稳定干扰HCT116细胞株。

Objective：To construct a recombinant vector of short hairpin RNA（shRNA） targeting TERF2IP1 gene,and to identify its inhibitory effect in HCT116 cells.Methods：the TERF2IP1 shRNA sequences were designed and inserted into pGPHI/Hygro vector.HCT116 cells were transiently transfect with shRNA vector and control vector,selected positive clone by hygromycin for 2~3 days.The total RNA and protein were extracted from the cells and the silence effect of TERF2IP1 was detected.Results：Four pair pGPHI/Hygro vector targeting TERF2IP1 were constructed,and the stably TERF2IP1 knockdown HCT116 cells were obtained.The expression of TERF2IP1 in HCT116 cells were downregulated significantly,confirmed by real-time PCR and Western blot.Conclusion：The constructed shRNA vector targeting human TERF2IP1 gene can stably suppress TERF2IP1 expression in HCT116 cells.