【摘要】 用分子克隆技术构建人gdnf基因打靶牛β-casein基因座的正负筛选打靶载体,用于制备生产人GDNF的牛乳腺生物反应器。打靶载体以牛β-casein基因上游和下游5.7 kb序列为5′和3′同源臂;neo抗性基因为正筛选因子;HSV-tk基因和DsRed2基因为双负筛选因子;人gdnf基因置于5′同源臂下游,SV40 polyA序列插入到人gdnf基因下游作为人gdnf基因转录终止信号。经PCR、限制性内切酶图谱及DNA测序分析鉴定,结果表明已成功地构建了人gdnf基因打靶牛β-casein基因座的正负筛选打靶载体,为研究人gdnf基因在牛β-casein基因座的定点整合及通过体细胞核移植法制备人GDNF牛乳腺生物反应器的研究奠定了基础。更多还原

【Abstract】 The goal of this study was to construct targeting vector for the human GDNF cDNA knock-in the cattle beta-casein gene locus so that human GDNF protein might be produced in gene-targeted cattle mammary gland.The pPGK-neoLoxP vector was used as plasmid backbone to construct the targeting vector pNRTCNbG.The 5′ and 3′ homologous arms as the beta-casein gene fragments were amplified from purified genomic DNA of the cattle by PCR.The human GDNF cDNA amplified by RT-PCR was located at the downstream of the 5′ arm.Moreover,SV40 polyA signals sequence was located at the downstream of the human GDNF gene as transcriptional ending signals.The neo gene was located between the 5′ and 3′ homologous arms.The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes,respectively.The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing.The results showed that the structure of the final constructed vector accords with the designed plasmid map.更多还原