摘要
为探讨绵羊内源性反转录病毒(enJSRV)在绵羊胚胎发育过程中的作用,利用脂质体将pEGFP-C1空载体转入绵羊绒毛膜滋养层细胞中,测定其转染效率。同时构建抑制enJSRV env基因的RNA干扰质粒,并将其转染到能稳定表达enJSRV env基因的绵羊绒毛膜滋养层细胞中,通过荧光定量PCR检测其干扰效率。结果显示,重组干扰质粒enJRSV-env-shRNA-1、enJRSV-env-shRNA-2、enJRSV-env-shRNA-3、enJRSV-env-shRNA-4、Negative-shRNA构建成功。Lipofectamine LTX脂质体体积为6.0μL,转染混合物体积为100μL,转染48h组的转染效率最高,为42.37%。与正常对照组细胞的enJSRV-env mRNA表达量相比,转染靶基因干扰质粒的各组绵羊绒毛膜滋养层细胞中enJSRV-env mRNA表达量分别下调了73.56%、66.82%、33.66%和18.16%。筛选出了一个干扰效率最高的RNA干扰质粒enJRSV-env-shR-NA-1,为以后包装慢病毒及进行动物试验来揭示enJSRV的生殖生物学作用奠定了坚实的基础。
The aim of this study was to investigate the biological role of sheep JSRV-related endogenous betaretroviruses（enJSRVs） in conceptus（embryo and associated extraembryonic membranes） development.Lipofectamine LTX was used as transfection vector for pEGFP-C1 plasmid,which carries report gene,enhanced green fluorescent protein（EGFP）,to transfect sheep trophoblast cells;and transfection efficacy was determined.Four enJRSV-env small interfering RNA（siRNA） sequences and one negative control siRNA sequence were subcloned respectively into the green fluorescence eukaryotic expression plasmid（pcDNA6.2-GW/EmGFP-miR） containing enJRSV-env siRNA gene.These five chimeras expected to inhibit enJRSV-env expression were respectively transfected into sheep trophoblast cells for stably expres-sing enJRSV-env.The fluorescent quantitative PCR was used to test the copies of enJRSV-env gene.The results showed that the five chimeras enJRSV-env-shRNA-1~4 and Negative-shRNA were successfully constructed.When used 6 μL Lipofectamine LTX,100 μL transfection mixture and 48 h post-transfection,the highest transfection efficiency of sheep trophoblast cells was obtained at 42.37%.The enJRSV-envexpression in sheep trophoblast cells transfected with 4 above enJRSV-env-shRNA chimeras were suppressed significantly,especially in the cells with enJRSV-env-shRNA-1 whose inhibition ratio reached 73.56%.This study provided insights into enJSRV influences on reproductive system and good preparation for lentivirus packaging.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第1期53-59,共7页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(31160493
30960271)
内蒙古科技创新引导基金项目(20101808)
教育部博士点基金博导类项目(20111515110008)
