pEGFP-loxP-Lox2272载体的构建及牛胎儿转基因阳性细胞筛选程序的优化  

Construction of a Vector pEGFP-loxp-Lox2272 and Optimization of Procedure for Selecting Transgenic Bovine Fetal Fibroblasts

作　　者：王帅[1,2] 赵学明[2] 朱化彬[2] 杜卫华[2] 郝海生[2] 王栋[2] 刘岩[2] 秦彤[2] 王宗礼[1] 

机构地区：[1]甘肃农业大学动物科学技术学院,兰州730070 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2011年第4期711-718,共8页Journal of Agricultural Biotechnology

基　　金：国家奶牛产业技术体系项目(No.nycytx-10);国家转基因专项(No.2009ZX08010-015B;NO.2009ZX08011-031B)共同资助

摘　　要：绿色荧光蛋白(GFP)是一种分选细胞的理想标记,将绿色荧光蛋白基因定点整合到牛的Y染色体上是利用GFP分选X和Y精子的关键。本研究通过特殊的引物设计和高保真酶PCR扩增将loxp位点和Lox2272位点引入pEGFP-N3载体中,构建了重组质粒pEGFP-loxp-Lox2272。采用LipofectamineTM(Lip)2000介导线性化质粒pEGFP-loxp-Lox2272转染牛胎儿成纤维细胞,结果显示,在质粒用量一定的情况下30μLLip2000转染细胞的效果最理想;转染后的牛胎儿成纤维细胞用G418筛选,结果显示,浓度为600μg/mL的G418最适合筛选转基因细胞。本实验的结果为载体改造提供了一种有效的方法并为牛体细胞基因打靶阳性细胞系的筛选提供了重要的技术参考。Green fluorescent protein（GFP） is an ideal marker for cell sorting.Integrating green fluorescent protein（GFP） into bovine Y chromosome is the key for sorting X and Y sperm by GFP.In this study,one loxp site and one Lox2272 site were inserted into the vector pEGPP-N3 through special designed primers amplified by high-fidelity enzyme and the vector pEGFP-loxp-Lox2272 was constructed.The bovine fetal fibroblasts were transfected with linearized pEGFP-loxp-Lox2272 by LipofectamineTM（Lip） 2000.The results revealed that the best transfected effect was mediaed by 3 μL Lip 2000 and the best concentration of G418 for screening transgenic cell clones was 600 μg/mL.This study provids a useful method for constructing vectors and important reference for screening bovine gene-targeted cell clones.

关 键 词：绿色荧光蛋白 阳性细胞筛选 基因打靶 载体构建 

分 类 号：Q785[生物学—分子生物学]