| 曾思聪周菂卢光琇.端粒酶shRNA 慢病毒载体的构建及转染人类胚胎干细胞[J].,2011,11(8):1401-1403 |
| 端粒酶shRNA 慢病毒载体的构建及转染人类胚胎干细胞 |
| Construction of telomerase shRNA lentivirus vector and transfectionof human embryonic stem cells |
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| DOI： |
| 中文关键词: 人类胚胎干细胞，慢病毒载体，转染 |
| 英文关键词: Human embryonic stemcells Lenti-virusvector Transfection |
| 基金项目:国家重点基础研究发展计划（973 计划）(2007CB948103)；中国高技术发展研究计划（863 计划）(2006AA02A102)；
湖南省科技计划项目(2008FJ3133) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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