利用逆转录病毒载体建立SMAD2稳定干扰的人胚胎干细胞系

Establishing SMAD2-Knocked down Human Embryonic Stem Cells by ShRNA Retroviral Vector

目的:建立SMAD2稳定干扰的人胚胎干细胞系。方法:利用包装细胞获得重组的逆转录病毒,感染人类胚胎干细胞,为干扰组ShSMAD2、载体组VECTOR和野生型组WT;经荧光筛选获得阳性克隆;Realtime-PCR检测SMAD2 mRNA的表达。结果:带GFP荧光标记的SMAD2特异性shRNA逆转录病毒感染人类胚胎干细胞后,获得稳定干扰SMAD2表达的人胚胎干细胞系。经检测shSMAD2组SMAD2 mRNA表达较VECTOR和WT组明显降低,VECTOR和WT组之间无明显差异。结论:通过SMAD2特异性shRNA逆转录病毒载体构建了稳定干扰SMAD2的人类胚胎干细胞。

Objective： To establish stably SMAD2 shRNA interfered human embryonic stem cell line.Methods： SMAD2 shRNA expressed pRetroSuper-GFP-SMAD2 vector was made virus in packaging cell line.Human embryonic stem cells were infected,which were divided into interference group（ShSMAD2）,VECTOR group and wild-type group（WT）.Positive clones were selected by GFP Fluorescence.SMAD2 knocked down efficiency were evaluated by Real-time PCR analysis.Results： After human embryonic stem cells were infected with SMAD2-specific shRNA retrovirus labeled GFP fluorescence,stable interference of SMAD2 expression in human embryonic stem cell lines had been obtained,in which SMAD2 mRNA expression was significantly lower compared with the VECTOR and the WT group,while VECTOR group and WT group showed no significant difference.Conclusion： Retroviral vector-delivered shRNA resulted in effective and stable down-regulation of SMAD2 expression in human embryonic stem cells.