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

农业生物技术学报，2011 年，第19 卷，第4 期，第711~718 页 Journal of Agricultural Biotechnology, 2011, Vol.19, No.4, 711~718 研究报告 A Letter 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 1，2 2 2 2 2 2 2 2 1* 王帅 赵学明 朱化彬 杜卫华 郝海生 王栋 刘岩 秦彤 王宗礼 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 * 通讯作者，wangzongli@ 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30 μL Lip 2000 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600 μg/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。 关键词 绿色荧光蛋白，阳性细胞筛选，基因打靶，载体构建 Construction of a Vector pEGFP-loxp-Lox2272 and Optimization of Procedure for Selecting Transgenic Bovine Fetal Fibroblasts Wang Shuai1,2 Zhao Xueming2 Zhu Huabin2 Du Weihua2 Hao Haisheng2 Wang Dong2 Liu Yan2 Qin Tong2 Wang Zongli1* 1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China * Corresponding author, wangzongli@ DOI: 10.3969/j.issn.1674-7968.2011.04.017 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 3 μL Lip 2000 and the best concentrat