绵羊Myostatin基因敲除载体的构建

Construction of Sheep Myostatin Gene Targeting Vector

根据Myostatin基因突变可导致肌肉量激增而产生"双肌"表型的特点,构建绵羊Myostatin基因置换型敲除载体。利用LA-PCR技术成功地扩增得到绵羊Myostatin基因同源臂序列,其中同源长臂4.9 kb,包括全部的exon1,intron1,exon2及部分启动子和大部分intron2;同源短臂1.1 kb,包括部分exon3和3′非翻译区序列,将二者连入PloxpII正负筛选敲除骨架载体,利用骨架载体上Neo基因替代Myostatin基因的exon3,从而成功构建专门针对Myostatin第3外显子区域缺失的置换型敲除载体PloxpⅡ-OVIS-MSTN。酶切和测序鉴定证明载体构建正确,为后续获得绵羊Myostatin基因缺失型体细胞株奠定试验基础。

Sheep myostatin gene replacement type knock-out vector was constructed,according to myostatin mutations that lead to muscle quantity surge to produce ＂double muscle＂ phenotype characteristics.In this study,sheep myostatin gene homologous arm sequence was successfully amplified in using LA-PCR technology;the 4.9 kb homologous long arm contained the whole exon1,intron1,exon2,parts of the promoter and most of the intron2;while the 1.1 kb homologous short arm contained the partial exon3 and 3′UTR sequence.The myostatin gene was replaced by neomycin phosphotransferase gene after the homologous arms cloned into PloxpII vector,for inactivating their function and achieving the knock-out purpose.PloxpII-OVIS-MSTN,which was replacement type knock-out vector specifically to deletion of exon3 of myostatin gene proved to be correct by restricted endonucleases digestion and sequence analysis.That laid the foundation for obtaining the myostatin gene deletion sheep somatic cell line.