【摘要】 目的构建小鼠Wnt-5a逆转录病毒载体,观察Wnt-5a在3T3细胞的表达情况。方法采用同源重组技术将小鼠Wnt-5a插入逆转录病毒载体PMX-IRES-GFP,构建PMX-IRES-Wnt5a重组质粒,利用脂质体转染3T3细胞,RT-PCR检测Wnt-5a mRNA表达情况,Western-Bloting检测Wnt-5a蛋白表达情况。结果经限制性内切酶证实成功构建了携带Wnt-5a基因的逆转录病毒载体PMX-IRES-Wnt5a,RT-PCR检测结果显示Wnt-5a在转染的3T3细胞的表达明显增高。结论成功构建Wnt-5a逆转录病毒载体,并能在3T3细胞成功表达Wnt-5a。更多还原

【Abstract】 Objective To construct the retroviral expression vector of PMX-IRES-Wnt5a,transfect into 3T3 cells,and observe the expression of Wnt-5a.Methods Gene recombinant technology was employed to clone mouse Wnt-5a gene to the retroviral expression vector of PMX-IRES-GFP,to obtain the recombined plasmid PMX-IRES-Wnt5a,transfect into 3T3 cells by Lipofectamine 2000.The result of transfection was analyzed by RT-PCR and Western-Blotting.Results Identification by enzyme digestion confirmed successful construction of the retroviral expression vector of PMX-IRES-Wnt5a.RT-PCR revealed that the expression of Wnt-5a mRNA and protein was obviously increased in 3T3 cells after transfection.Conclusion The retroviral expression vector of PMX-IRES-Wnt5a is successfully constructed and stably expressed in 3T3 cells.更多还原