绵羊抑制素shRNA慢病毒干扰载体的构建与鉴定

Construction and Identification of a Lentiviral Vector Expressing shRNA for RNA Interference Sheep Inhibin Alpha Subunit(INHA)

构建并鉴定了针对绵羊抑制素α亚基(INHA)基因的shRNA(Small hairpin RNA,shRNA)慢病毒干扰载体,为进一步制备优质、高产转基因绵羊奠定基础。设计并合成4条针对绵羊INHA基因的shRNA表达序列(shRNA1、shRNA2、shRNA3、shRNA4)及2条阴性对照序列(NC1、NC2),将其分别连接到空载体pGFP-V-RS中,得到4个shRNA表达载体(即shRNA干扰载体)pGFP-V-RS-shRNA和2个阴性对照载体pGFP-V-RS-NC;同时构建绵羊INHA的高效表达载体pIRES2-eGFP-INHA,然后将构建好的4个shRNA干扰载体及2个阴性对照载体分别与INHA高效表达载体瞬时共转染293T细胞,qPCR法进行干扰效果筛选,挑选干扰效果最佳的shRNA干扰载体构建成慢病毒干扰载体。qPCR检测结果表明,shRNA干扰载体pGFP-V-RS-shRNA3的干扰效果最好,用它构建的慢病毒干扰载体经测序验证表明其已建成功。绵羊INHA shRNA慢病毒干扰载体构建成功,为进一步制备优质高繁转基因绵羊奠定了基础。

To construct a lentiviral vector expressing shRNA for RNA interference of sheep inhibin alpha subunit（INHA） gene and assess its gene silencing effect in 293T cell by cotransfected with an efficiently expressional INHA vector constructed simultaneously.Make preparation for producing high quality and high fecundity transgenic sheep.Design and synthesis 4 shRNA expressing sequences（shRNA1,shRNA2,shRNA3,shRNA4）and 2 control sequences（NC1,NC2）for sheep INHA gene,and then attach them to a blank plasmid vector： pGFP-v-RS,thus 4 shRNA expressing vector（that is shRNA interfering vector） pGFP-V-RS-shRNAs and 2 negative control vector pGFP-V-RS-NCs were generated;At the same time,construct an efficiently expressional vector of sheep INHA pIRES2-eGFP-INHA,then cotransfect 4 shRNA interfering vectors and 2 negative control vectors with efficiently expressional INHA vectors into 293T cell respectively and instantaneously,assess the interference effect of 4 shRNA interfering vectors contrasting with/in comparison to the 2 negative control vectors by qPCR,choose the shRNA interfering vector with the best interference effect,and construct it into corresponding lentiviral vector.The qPCR results indicate that the shRNA interference vector pGFP-V-RS-shRNA3 has the best interference effect,and the lentiviral vector constructed by it has been proved to be successfully constructed by sequencing results.Lentiviral interference vector for sheep INHA shRNA has been constructed successfully,and make the preparation for furtherly producing high quality and high fecundity transgenic sheep.