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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
