Ｌｉｖｉｎ特异性ｓｉＲＮＡ表达载体在胃癌细胞中的稳定表达

目的:构建以Livin基因为靶基因的siRNA表达载体pGPU6/GFP/Neo/Livin,获得稳定沉默Livin基因的胃癌细胞SGC-7901/siRNA-Livin.方法:化学合成siRNA,Livin寡核苷酸,将其分别克隆进载体pGPU6/GFP/Neo,进行核酸序列测定.脂质体法转染SGC-7901细胞,半定量RT-PCR和Western blot检测Livin基因在SGC-7901细胞中的沉默效率,G418筛选获得稳定转染的细胞克隆.结果:酶切分析显示,所有质粒均为阳性重组载体,核酸测序表明合成的序列准确插入pGPU6/GFP/Neo载体,G418筛选基因转染SGC-7901细胞后形成稳定克隆.其中pGPU6/GFP/Neo/Livin-4转染后Livin mRNA沉默效率大于70%.结论:成功获得Livin特异性siRNA表达载体,SGC-7901细胞Livin基因的表达受到抑制,转染胃癌细胞后获得SGC-7901/siRNA-Livin细胞克隆.

Construction of Livin specific siRNA expression vector and its stable expression in SGC-7901 cells

Objective:To establish Livin specific gene-silencing system and to obtain stable clones of SGC-7901/siRNA-Livin. Methods:The small interfering RNA eukaryotic expression vector specific to Livin was constructed by gene recombination,and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot were used to validate gene silencing efficiency of Livin in SGC-7901 cells. Stable clones were obtained by G418 screening. Results:Restriction endonuclease analyses showed that all plasmids had been successfully recombined. Nucleic acid sequencing showed that the siRNA was synthesized and inserted into pGPU6/GFP/Neo exactly. Livin silenced SGC-7901 cell clones were obtained after gene transfection and G418 selection,while the silencing efficiency of pGPU6/GFP/Neo/Livin-4 was higher than 70%. Conclusion:Livin specific siRNA was successfully constructed,and the expressions of Livin was silenced. Stable clones were obtained after gene transfection and screening.