猪MSTN基因敲除载体的构建及细胞筛选  

Construction of MSTN knock-out porcine vector and MSTN knock-out porcine fetal fibroblast

作　　者：高飞[1] 唐成程[1] 全龙泉[1] 戴祯[1] 苏佰通[1] 赖良学[1] 逄大欣[1] 欧阳红生[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2013年第1期142-145,160,共5页Chinese Journal of Veterinary Science

基　　金：转基因生物新品种培育重大专项(2011ZX08006-003)

摘　　要：构建猪肌肉生长抑制素(Myostatin,MSTN)基因的打靶载体并获得敲除MSTN基因的猪胎儿成纤维细胞。以Puro为正筛选基因,白喉毒素-A(DT-A)为负筛选基因。将同源长臂和同源短臂分别插入Puro基因的两侧。同源长短臂分别为4 294bp和1 015bp,定点敲除MSTN基因的部分内含子2和部分外显子3。采用FugeneHD转染法将打靶载体转入37d的猪胎儿成纤维细胞中,转染后的细胞采用嘌呤霉素筛选。结果显示,成功构建了对猪MSTN基因部分区域进行敲除的打靶载体,共得到48个具有药物抗性的细胞克隆,经PCR检测,获得2个正确同源重组的细胞克隆。The objective of this study is to construct a targeting vectors for knocking-out porcine MSTN gene and get MSTN knock-out porcine fetal fibroblast.Construction of targeting vector：puromycin gene was used as positive-selecting gene,diphtheria toxin gene A fragment was used as negative-selecting gene.Homologous arms were inserted into the two sides of puromycin gene,respectively.The homologous arms are 4 294 bp and 1 015 bp which can knock out partial intron2 and exon3 of myostatin.Targeting vector was introduced into the 37 d porcine fetal fibroblast by FugeneHD transfection methods,after transfection,selected with puromycin.PCR analysis of clones was done using the lysate of pig fetal fibroblast after transfection.The vector was successfully constructed,which can knock out partial intron2 and exon3 of myostatin.Forty eights resistance cell strains were obtained,two strains took place correct homologous recombination through PCR identification.

关 键 词：猪 MSTN基因 基因敲除 打靶载体 

分 类 号：Q78[生物学—分子生物学] S814[农业科学—畜牧学]