山羊体细胞转基因方法的探讨  

Research of Transfection Method of Fetal Fibroblasts in the Goat

作　　者：潘求真[1] 陆泉枝[2] 王海[2] 敖红[3] 连正兴[2] 李宁 吴常信[2] 

机构地区：[1]中国农业大学动物科技学院 [2]中国农业大学动物科技学院农业部畜禽遗传育种重点实验室,北京100094 [3]农业生物技术国家重点实验室 [4]农业生物技术国家重点实验室农业部畜禽遗传育种重点实验室,北京100094

出　　处：《畜牧兽医学报》2004年第2期121-124,共4页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金资助项目(30170675);"863"计划(2001AA213031)。

摘　　要：转基因体细胞核移植是制备高效表达转基因动物的主要手段与方法,但由于插入位点、拷贝数不同常使外源基因的表达受到一定影响。本试验以双正向选择标记的pLoxP M为转基因结构,以磷酸钙法、脂质体介导法、电击转染三种方法对山羊胎儿成纤维细胞进行转染。电击(1 70×10-4)和脂质体(1 36×10-4)法明显高于磷酸氢钙法(4 6×10-5),而且三方法的转染效率与细胞类型无关,但电击转染方法简单并适合于不同大小的转基因结构,对长片段更为实用。随机整合的转基因细胞系绿色荧光蛋白的表达水平有很大差异,主要与外源基因的拷贝数和插入位点有关。双正向选择标记不但可以筛选重组体而且可以体外监控外源基因的表达情况,为高表达转基因动物的制备提供了良好的手段与方法。The nuclear transfer of transgenic somatic cell is an effective method for making transgenic animal with efficient expression of foreign gene.Transgenic structure of pLoxP- M plasmide was built which contained double positive selection marker genes(noe^r and GFP),double homologous arms and TK as negative selection marker in this experiment.Three methods of CaHPO4,lipofectamin and electroporation were used to transfect the fetal fibroblasts with the transfection efficiency of 4.6 ×10^(-5),1.36×10^(-4) and 1.70×10^(-4) respectively.No significant difference appeared between fetal and adult fibroblasts in transfection efficiency.The electroporation was the best method for large fragmnet of transgenic structure.The level of GFP gene expression showed significant difference among different transgenic cell lines integrated randomly,due to the difference of inserting loci and copy number of GFP gene.Transgenic vector with double positive selection markers was a good method to make transgenic animal with high level expression of foreign gene,because which not only could select recombinant,but also showed the expression of foreign gene.

关 键 词：山羊 体细胞 转基因方法 胎儿成纤维细胞 细胞核移植 

分 类 号：S827[农业科学—畜牧学]