The aim of the study was to knock out the MSTN gene in sheep, and adenoviral vector encoding zinc-finger nucleases (ZFN) targeted to the exon 1 of MSTN was constructed and packaged. Based on the high transfection efficiency and non-integration of the adenovirus combinding with the specificity and high performance of the ZFN, disruption of the MSTN in sheep was expected in the further research. DNA sequences of T2A and the two designed ZFNs monomers were amplified by PCR and inserted into the pAdTrack-CMV successively. The positive ones got were named as pAdTrack-ZFNL-T2A-ZFNR. Then PAdTrack-ZFNL-T2A-ZFNR and pAdEasy-1 were co-transfected into E. coli. BJ5183 in which homologous recombination would happen between the two plasmids, the recombinant adenoviral vector was named as pAdEasy-ZFNL-T2A-ZFNR. The recombinant vector was transfected into the HEK293 subsequently to produce the final adenovirus. Finally, the adenovirus was identified by PCR and the titer was detected, , then the positive ones were reduplicated. The final adenovirus were added to the culture medium of sheep fetus fibroblasts to further confirm the infectivity of the virus and identify the activity of ZFN in mammalian cells by Western blot. The adenoviral vector containing MSTN-ZFN expres-sion cassette was verified by enzymatic digestion and sequencing, and ZFN could recognize and cleave the target site of MSTN. The adenoviral vector encoding ZFN could target to the MSTN in sheep, and it was constructed successfully.%旨在构建并包装打靶绵羊MSTN基因的位点特异性锌指核酸酶腺病毒表达载体,以借助腺病毒的高转染效率和非整合性以及锌指核酸酶的高效性和特异性实现对绵羊MSTN基因的敲除.本研究利用PCR扩增T2A序列和锌指核酸酶异源二聚体序列,测序鉴定后依次克隆至pAdTrack -CMV,得到pAdTrack-ZFNL-T2A-ZFNR穿梭载体,将之与腺病毒骨架载体pAdEasy -1共转染至BJ5183菌株进行同源重组,以构建pAdEasy-ZFNL -T2A-ZFNR表达载体.然后用上述表达载体转染HEK293细胞进行重组腺病毒的包装,将PCR鉴定阳性的病毒进行扩增并测定病毒滴度.侵染绵羊胎儿成纤维细胞检测重组腺病毒对靶细胞的侵染能力,并用Western blot方法检测绵羊胎儿成纤维细胞中ZFN的表达,进而在细胞水平验证ZFN的活性.结果,包装得到的锌指核酸酶重组腺病毒能够高效侵染绵羊胎儿成纤维细胞并表达ZFN,腺病毒介导的ZFN可识别并切割绵羊MSTN基因.本研究成功获得靶向识别并切割绵羊MSTN基因的锌指核酸酶重组腺病毒.
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