小鼠微小RNA294逆转录病毒载体的构建及鉴定  

Construction and identification of embryonic stem cell-specific microRNA294 retroviral vector of mice

作　　者：许杰[1] 闫秋月[1] 湛彦强[1] 张苏明[1] 

机构地区：[1]华中科技大学附属同济医院神经内科,武汉430030

出　　处：《郑州大学学报（医学版）》2011年第1期19-22,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30370505;武汉市科技计划基金资助项目200860423216

摘　　要：目的:构建小鼠微小RNA294(miR-294)逆转录病毒载体。方法:从小鼠基因组DNA扩增pri-miR-294,克隆连接至pMX-IRES-GFP逆转录病毒载体上,得pMX-miR-294-IRES-GFP,测序鉴定后以转染PLAT-E细胞进行病毒包装,测定病毒滴度。结果:pMX-miR-294-IRES-GFP经PCR扩增及测序鉴定正确,并得到了滴度为(1～5)×106TU/mL的病毒液。结论:成功构建了pMX-miR-294-IRES-GFP逆转录病毒载体。Aim：To construct microRNA-294（miR-294）retroviral vector.Methods：pri-miR-294 amplified by PCR was inserted into pMX-IRES-GFP vector,and then identified by nucleotide sequencing.PLAT-E cells were transfected with retroviral vector pMX-miR-294-IRES-GFP.All virus stocks were produced by calcium phosphate-mediated transfection.The titer of virus was tested according to the expression level of GFP.Results：DNA sequencing demonstrated that pMX-miR-294-IRES-GFP was successfully constructed.The titer of concentrated virus was（1-5）×10^6 TU/mL.Conclusion：pMX-miR-294-IRES-GFP retroviral vector was successfully constructed,which paved the way for the induction of induced pluripotent stem cells and the investigation of cellular reprogramming mechanism.

关 键 词：微小RNA 细胞重编程 逆转录病毒载体 小鼠 

分 类 号：R394[医药卫生—医学遗传学]