【摘要】 针对蒙古绵羊肌肉生长抑制素(myostatin,MSTN)基因序列的第3外显子,选择设计并合成了含有限制性内切酶酶切位点及含有与敲除载体同源20bp碱基的2种引物。以蒙古绵羊的血液DNA为模板,利用PCR分别扩增出2套用于敲除载体的同源长短臂。将这2套同源长短臂分别连接到2个不同的基础载体pPNTⅢ和pEGFP-N1中,构建2套用于蒙古绵羊MSTN基因敲除的置换型打靶载体pPNTⅢ-S21-L32和pEGFP-N1-S0-L0,其中pPNTⅢ-S21-L32为含有正负筛选标记基因的传统敲除载体,pEGFP-N1-S0-L0为优化后正筛选标记基因为绿色荧光加强的敲除载体。经PCR、T载体连接、酶切鉴定和DNA测序证实,2套同源臂分别被正确插入到基础载体中。本研究构建的2套用于蒙古绵羊肌肉生长抑制素基因敲除的置换型打靶载体,为改善家畜产肉性状,增加肌肉质量奠定了基础。更多还原

【Abstract】 Based on sequencing and analysis of the Mongolian sheep myostatin gene sequence,specially for exon 3,we selected and designed two primers.One of the primers has a restricted endonucleases site,and another of the primers has 20 homologous bases,which were consistent with the knockout vector.Then the two types of long and short homologous arms for both knock-out vectors were isolated from genomic DNA of Mongolian sheep blood by PCR.Finally,these homologous fragments were cloned into two different basic targeting vectors,pPNTⅢ and pEGFP-N1,to construct two replacement targeting vectors,pPNTⅢ-S21-L32 and pEGFP-N1-S0-L0.The targeting vector pPNTⅢ-S21-L32 is a traditional knock-out vector with positive and negative selection marker genes,and the targeting vector pEGFP-N1-S0-L0 is a knockout vector with enhanced green fluorescence after optimizing.By PCR,T-vector ligation,restriction enzyme digestion and DNA sequencing,confirmed that the short and long arms were included in the two targeting vectors.The two types of constructed replacement targeting vectors for knock-out Mongolian sheep MSTN gene laid the foundation for the improvement of livestock meat production traits and increasing muscle mass.更多还原