农业生物技术学报2011，Vol.19Issue(4)：711-718,8.DOI:10.3969/j.issn.1674-7968.2011.04.017
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
摘要
Abstract
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 3μL Lip 2000 and the best concentration of G418 for screening transgenic cell clones was 600μg/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Key words
Green fluorescent protein/Selecting transgenic clone cells/Gene targeting/Vector construction引用本文复制引用
王帅,赵学明,朱化彬,杜卫华,郝海生,王栋,刘岩,秦彤,王宗礼..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[J].农业生物技术学报,2011,19(4):711-718,8.基金项目
本研究由国家奶牛产业技术体系项目(No.nycytx-l0)和国家转基因专项(No.2009ZX08010-015B （No.nycytx-l0）
NO.2009ZX08011-031B)共同资助 （）
