三位点短发卡状RNA在提高RNA干扰效率中的作用  

Effect of three different short hairpin RNAs in the enhancement of RNA interference

作　　者：翁艳洁[1] 翁丹卉[1] 夏曦[1] 宋晓红[1] 卢运萍[1] 王世宣[1] 马丁[1] 王常玉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,湖北武汉430030

出　　处：《中国癌症杂志》2008年第4期249-253,共5页China Oncology

基　　金：国家自然科学基金资助项目(编号:30571950);国家自然科学基金资助项目(编号:30528012);国家973重点基础科学基金资助项目(编号:2002CB513107)

摘　　要：背景与目的:shRNA技术是近来发展的热点技术之一,但普遍采用的单位点shRNA干扰载体效果并不理想。本研究探讨三位点短发卡状RNA在提高RNA干扰效率中的作用。方法:构建外源基因DsRed全长载体转染HEK293细胞。针对报告基因DsRed设计4个干扰载体,一个含有3段shRNA,另外3个分别含有此3段中1段shRNA。转染293/DsRed,荧光显微镜观察基因DsRed。应用实时定量PCR(Real-timePCR)和流式细胞仪(FACS)检测4个载体对DsRed基因在mRNA及蛋白水平干扰效应。结果:4个载体对靶基因DsRed在mRNA及蛋白水平均有干扰效应,且含有3段shRNA载体对DsRed基因干扰效率为(89±16)%,显著高于3个只含有1段shRNA载体[(50±23)%,(52±15)%,(55±27)%],差异有显著性(P<0.05)。结论:三位点shRNA干扰载体可以提高对靶基因的干扰效率。Background and purpose： shRNA skill is one of the hottest spots in the area of cancer biology recently, though its result is not very effective, It is possible that the interfering vector contain only one shRNA fragment. We investigated the efficacy of RNA interference by using three shRNAs. Methods： Vector of DsRed was designed and constructed, and transfected into HEK293 cells, cells with stably expressing vector has been obtained by screening. Four vectors targeting DsRed were designed and constructed, one with three different shRNAs and other three with just one of three shRNAs. All four vectors were transfeeted into HEK293/DsRed respectively. The expression of red fluorescence was evaluated by fluorescent microscope. Semiquantitative Real-time PCR and FACS were used to detect the efficiency of gene silencing of the four vectors. Results： All four vectors inhibited the expression of DsRed in mRNA and protein levels. The vector with three shRNAs had a significantly higher gene silencing efficiency than the other three that contained only one of these three shRNAs[ （ 89±13） % vs. （50±23） %, （ 52±15） % and （ 55±27） %, P 〈 0.05） ]. Conclusions： The vector containing three different shRNAs could produce a much better gene silencing effect.

关 键 词：RNA干扰 外源基因 DSRED 短发卡状RNA 

分 类 号：R730.2[医药卫生—肿瘤]