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绵羊抑制素shRNA慢病毒干扰载体的构建与鉴定

DOI: 10.7668/hbnxb.2011.03.005, PP. 21-27

Keywords: 抑制素&alpha,亚基,慢病毒,小发夹RNA,RNA干扰,绵羊

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Abstract:

构建并鉴定了针对绵羊抑制素α亚基(INHA)基因的shRNA(SmallhairpinRNA,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INHAshRNA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.

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