小鼠Dppa2基因shRNA载体的构建及干扰效果的检测  

Construction and Evaluation of shRNA Recombinant Plasmids Targeting Mouse Dppa2 Gene

作　　者：陈天姬[1] 杜娟[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,湖南长沙410078

出　　处：《现代生物医学进展》2010年第3期404-407,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(30570937);中南大学研究生创新基金资助项目(2340-74334000003)

摘　　要：目的:构建有效的针对小鼠Dppa2基因的shRNA(short hairpin RNA)干扰载体。方法:设计合成2对针对小鼠Dppa2基因的shRNA序列以及1对与哺乳动物基因组无同源性的shRNA序列作为对照,构建pSUPER.Retro.puro干扰载体并进行PCR,酶切和测序验证。进一步将各干扰载体分别转染小鼠胚胎干细胞(embryonic stem cells,ESCs),RT-PCR检测干扰效率。结果:PCR,酶切和测序验证均表明各shRNA载体构建成功。将空载体及各重组载体分别转染小鼠ESCs发现,干扰组Dppa2基因表达水平相对于空载体对照组和阴性shRNA载体对照组明显下调。结论:成功构建了有效的针对小鼠Dppa2基因的shRNA干扰载体,为进一步研究Dppa2基因在维持小鼠ESCs不分化过程中的作用提供了基础。Objective：To construct effective shRNA （short hairpin） recombinant plasmids targeting mouse Dppa2 gene. Methods： Two pairs of shRNA sequences targeting mouse Dppa2 gene and one pair of negative control shRNA sequence were designed and synthesized, and then inserted into the pSUPER.Retro.puro vector. The shRNA recombinant vectors were identified by PCR analysis, restriction enzyme digestion and sequencing, and were further transfected into mouse embryonic stem cells （ESCs）. Dppa2 knockdown efficiency were evaluated by RT-PCR analysis. Results：The results of PCR analysis, restriction enzyme digestion and sequencing showed that the shRNA vectors were constructed successfully. Dppa2 expression was dramatically downregulated in pSUPER / Dppa2 shRNA-transfected mouse ESCs. Conclusion：The shRNA interference vectors targeting mouse Dppa2 gene were successfully constructed, laying a foundation for further research of the Dppa2 function in maintaining the undifferentiated state of mouse ESCs.

关 键 词：Dppa2 RNA干扰(RNAinterference RNAi) shRNA载体 

分 类 号：Q953[生物学—动物学] Q291