小鼠MSTN shRNA可控表达载体构建及基因沉默效率研究  

Construction of controllable mice MSTN shRNA expression vector and the gene silencing efficiency study

作　　者：杨跃飞[1] 祁钰钰 杨开典 齐传翔 鞠辉明[1] YANG Yuefei QI Yuyu YANG Kaidian QI Chuanxiang JU Huiming(Coll of Vet Med, Yangzhou Univ, Yangzhou 225009, Chin)

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2016年第4期1-4,共4页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31101683;31272405);江苏高校品牌专业建设工程项目(PPZY2015B158);江苏省动物重要疫病与人兽共患病防控协同创新中心项目(2016-10)

摘　　要：肌肉生成抑制素(MSTN)是肌肉发育的负调控因子。根据MSTN mRNA序列设计1对MSTN shRNA并构建整合型四环素诱导表达MSTN shRNA载体pSingle-tTS-MSTN shRNA-GFP。重组载体转染小鼠成肌细胞并经G418筛选,通过强力霉素(DOX)诱导细胞中MSTN shRNA表达,利用荧光显微镜观察细胞中GFP表达情况,利用实时荧光定量PCR(QRT-PCR)及Western blot检测MSTN mRNA及蛋白的表达水平。结果表明:经DOX诱导后,转染含MSTN shRNA重组载体细胞组MSTN mRNA相对表达水平比未经DOX诱导组及其他对照组细胞中降低70%左右,差异极显著(P<0.01),MSTN蛋白也下降90%左右,其他各组间MSTN mRNA及蛋白表达量差异不显著(P>0.05)。该研究成功构建了可控表达MSTN shRNA载体,并在细胞水平证明其有很好的基因表达及调控效率,为后续制备可控表达MSTN shRNA转基因小鼠,通过精细调控MSTN基因的表达为研究该基因在肌肉发育中的功能奠定基础。Myostatin（MSTN）,previously referred to as growth differentiation factor 8,is a negative regulator of skeletal muscle growth.In this study,apair of MSTN shRNA was designed based on the MSTN sequence,and an integrated tetracycline induced expression vector,pSingle-tTS-MSTN shRNA-GFP,was constructed to express MSTN shRNA.The recombinant vector was transfected to C2C12 cell lines and screened with G418.MSTN shRNA expression was induced with DOX.The GFP expression was observed under a fluorescent microscope and the MSTN mRNA and protein were detected with QRT-PCR and western blot methods.The results showed that,after DOX inducing,the GFP can be observed and the MSTN mRNA decreased 70%in the cells groups transfected with pSingle-tTS-MSTN shRNA-GFP vector cell groups,the difference between them is extremely significant（P〈0.01）.The MSTN protein in this group decreased 90% or so,whereas the other 3groups showed no obvious difference（P〉0.05）.A controllable MSTN shRNA overexpression vector was constructed successfully and showed a well-controlled gene expression efficiency.This study lays the foundation for prepairing MSTN shRNA controllable expression transgenic mice and the gene function analysis on muscle development.

关 键 词：肌肉生成抑制素 可控表达 SHRNA 基因沉默 

分 类 号：O786[理学—晶体学]