同源重组敲除MSTN基因的猪胎儿成纤维细胞的构建  

Construction of MSTN Knock-out Porcine Fetal Fibroblast

作　　者：李景芬[1] 于浩[2] 袁野 刘娣 

机构地区：[1]湖州师范学院生命科学学院,浙江湖州313000 [2]吉林大学农学部畜牧兽医学院,长春130062 [3]公安部警犬技术学校警犬技术系,沈阳110034 [4]黑龙江省农业科学院,哈尔滨150086

出　　处：《中国农业科学》2009年第8期2972-2977,共6页Scientia Agricultura Sinica

基　　金：黑龙江省攻关项目(GB01B104)

摘　　要：【目的】获得敲除肌肉生长抑制素(MSTN)基因的猪胎儿成纤维细胞。【方法】打靶载体的构建:以Neo为正筛选基因、HSV-tk为负筛选基因。在Neo的两侧分别插入同源长臂和同源短臂。同源长臂5382bp,包含MSTN基因的部分5′端,全部的exon1,intron1和exon2及大部分intron2;同源短臂844bp,包含部分exon3及3′端的部分序列。取35d胎龄的大白猪,用胰酶消化法,分离胎儿成纤维细胞并对其进行培养和建系。采用脂质体法将打靶载体导入胎儿成纤维细胞中,转染后的细胞采用250μg·ml-1G418筛选7d,再用200μg·ml-1G418+2μmol·L-1GANC维持筛选。用RT-PCR法检测转染前转染后细胞MSTN基因表达量。【结果】成功构建了对猪MSTN基因部分intron2和exon3区域进行敲除的替代型打靶载体。共得到5个具有药物抗性的细胞克隆,经PCR检测,其中一个细胞克隆发生了正确的同源重组。转染后细胞MSTN基因表达量明显降低。【结论】获得了敲除MSTN基因的猪胎儿成纤维细胞。[Objective] The objective of this study is to get MSTN knock-out porcine fetal fibroblast. [Method] Construction of targeting vector： Neomycin phosphotransferase gene was used as positive-selecting gene, herpes simplex thymidine kinase gene was used as negative-selecting gene. Homologous arms were respectively inserted into the two sides of Neo gene. The 5.3 kb homologous left arm contains partial 5＇ MSTN gene, the whole exonl and exon2, and part of intron2, while the 0.8 kb homologous right arm contains part of exon3 and partial 3＇ MSTN gene. Taking the tissue of 35 days Yorkshire as materials, fibroblast lines were successfully established by trypsin digestion. Targeting vector was introduced into the fetal fibroblast of Yorkshire by lipofectamin-mediated DNA transfection methods, after transfection, selected with G418 （250 μg·ml^-1 ） for 7 days, then continuous selected with 200 μg·ml^-1 G418＋2 μmol·L^-1 GANC. Semi-quantitative RT-PCR analysis of MSTN mRNA expression was doneusing the total RNAs from pig fetal fibroblast before and after transfection. [Result] The replacement vector was successfully constructed, which can knock out partial intron2 and exon3 of myostatin. Five resistance cell strains were gotten, through PCR identification, one strain took place correct homologous recombination. The amount of MSTN mRNA expression of transgenic cells is obviously declined. [ Conclusion ] MSTN knock-out porcine fetal fibroblast was obtained.

关 键 词：猪 胎儿成纤维细胞 基因打靶 肌肉生长抑制素 

分 类 号：S828[农业科学—畜牧学]