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。
Objective To construct the retroviral expression vector of PMX-IRES-Wnt5a,transfect into 3T3 cells,and observe the expression of Wnt-5a（nethods Gene recombinant technology was employed to clone mouse Wnt-5a gene to the retroviral expression vector of PMpX-IRES-GFP, to obtain the recombined plasmid PMX-IRES-WntSa,transfect into 3T3 cells by Lipofectamine 2000. The result of transfection was analyzed by RT-PCR and Western-Blotting. Results Identifica tion by enzyme digestion confirmed successful construction of the retroviral expression vector of PMX-IRES-Wnt5a. RT-PCR revealed that the expression of Wnt-Sa mRNA and protein was obviously increased in 3T3 cells after transfection. Conclusion The retiroviral expression vector of PMX-IRES-WntSa is successfully constructed and stably expressed in 3T3 cells.
出处
《中国现代医生》
2012年第25期7-8,10,共3页
China Modern Doctor
基金
浙江省温州市2009年第一期科技计划项目(Y20090097)
