文章摘要
丁清清,刘 平.Livin特异性siRNA表达载体在胃癌细胞中的稳定表达[J].南京医科大学学报,2008,28(4):429~433
Livin特异性siRNA表达载体在胃癌细胞中的稳定表达
Construction of Livin specific siRNA expression vector and its stable expression in SGC-7901 cells
投稿时间:2007-11-03  
DOI:10.7655
中文关键词: Livin  RNA干扰  SGC-7901细胞  基因
英文关键词: Livin  RNA interference  SGC-7901  gene
基金项目:江苏省医学重点人才“135”工程资助项目(52-2001)
作者单位
丁清清 南京医科大学第一附属医院肿瘤科江苏 南京 210029 
刘 平  
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中文摘要:
      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。
英文摘要:
      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.
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