Oct4、Sox2、c-Myc、Klf4基因慢病毒载体的构建

【摘要】 目的构建Oct4、Sox2、c-Myc、Klf4基因慢病毒表达载体,为诱导性多能干细胞研究奠定基础。方法将Oct4、Sox2、c-Myc、Klf4基因利用Infusion技术重组构建慢病毒载体穿梭质粒pGC-FU-Oct4、pGC-FU-Sox2、pGC-FU-c-Myc、pGC-FU-Klf4,经PCR和测序后与与结构质粒pHelper1.0和包膜质粒pHelper2.0在脂质体Lipofectamine2000介导下共转染293T细胞包装生产慢病毒,浓缩纯化后检测滴度。结果 PCR扩增和测序结果证实成功构建Oct4、Sox2、c-Myc、Klf4的慢病毒载体并包装慢病毒,检测Oct4基因重组慢病毒、Sox2基因重组慢病毒、c-Myc基因重组慢病毒的滴度均为1×109TU/mL,Klf4基因重组慢病毒的滴度为1.9×109TU/mL。结论成功建立重组慢病毒载体的三质粒包装细胞系统。更多还原

【Abstract】 Objective To construct lentiviral vectors carrying Oct4,Sox2,c-Myc,Klf4 gene and provid a basis for further study on the induced pluripotent stem cells.Methods Oct4,Sox2,c-Myc and Klf4 genes were recombined to construct the transfer plasmids pGC-FU-Oct4,pGC-FU-Sox2,pGC-FU-c-Myc and pGC-FU-Klf4 by infusion technique.The 293T cells were cotransfected with the transfer plasmid,the construction plasmid Helper 1.0 and the envelope plasmid Helper 2.0 with the help of lipofectamine 2000 to produce lentiviral particles.Results PCR and DNA sequencing demonstrated that the in serted sequences were correct.The titer of Oct4,Sox2,c-Myc genes virus all were1.9×109 TU/ml, The titer of Klf4 gene virus was 1.9×109 TU/ml.Conclusion The three-plasmid packaging cell line system of recombinant lentiviral vector was successfully established.更多还原