端粒酶shRNA慢病毒载体的构建及转染人类胚胎干细胞

目的：构建端粒酶shRNA慢病毒载体及建立端粒酶稳定干扰的人类胚胎干细胞系。方法：将端粒酶基因特异性shRNA靶序列与慢病毒载体PLKO.1-puro连接、转化、挑取阳性克隆进行PCR及测序鉴定;利用包装细胞293T获得重组的慢病毒,感染人类胚胎干细胞,分为干扰组ShTert、载体组vector和野生型组wt;Realtime-PCR检测端粒酶mRNA的表达。结果：经PCR和DNA测序鉴定,成功构建端粒酶特异性shRNA慢病毒载体,并感染人类胚胎干细胞;经检测shTert组端粒酶mRNA表达较vector和wt组明显降低,vector组和wt组之间无明显差异。结论：通过成功构建的端粒酶特异性shRNA慢病毒载体对人类胚胎干细胞的转染实现了对其端粒酶mRNA的调控。

Construction of telomerase shRNA lentivirus vector and transfection of human embryonic stem cells

Objective： To construct telomerase shRNA lentivirus vector and the human embryonic stem cell system stably interfered by telomerase.Methods： Telomerase gene-specific shRNA＇s Target sequence and PLKO.1-puro lentiviral vector were connected and transformed.The positive clones were selected and the shRNA constructor was identified by PCR reaction and DNA sequencing.The recombinant lentivirus was obtained by packaging cells 293T.Human embryonic stem cells were infected,divided into interference group（ShTert）,vector group（vector） and wild-type group（Wt）.Expression of telomerase mRNA was examined by Realtime-PCR.Result： PCR and DNA sequencing showed that the telomerase-specific shRNA lentiviral vector was constructed successfully and infected in human embryonic stem cells.After testing,expression of telomerase mRNA in shTert group was significantly lower than that in vector and wt groups.There wasn＇t significant difference between vector and wt group.Conclusion： The telomerase shRNA lentivirus vector has been successfully constructed and human embryonic stem cells have been transfected and finally the regulation of telomerase mRNA has been achieved.