PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测.pdfVIP

中国农业科学 2012,45(17):3576-3583 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2012.17.014 PiggyBac 转座子介导的诱导细胞永生化转基因 载体的构建及其整合效率的检测 张婷婷, 辛 颖, 张志强, 任充华, 杨涵江, 王 令, 张智英 （西北农林科技大学动物科技学院基因组学实验室，陕西杨凌712100 ） 摘要： 【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】 以载体为模板，分别扩增 SV40P neo、IRES、tk-PloyA，通过 overlap PCR 连接，并在两端添加方向相同的 LoxP 序列，构建转基因基本载体PB-NIT；然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA 基因表达 盒，将其插入PB-NIT中，构建载体PB-NIT-STP；最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP， 构建载体 PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定，以上载体均正确；经转座活性鉴定，共转染转座子 载体 PB-rtTA-NIT-STP 和转座酶载体比只转染转座子载体获得的阳性克隆数提高了 20 倍。【结论】得到了基于 PiggyBac 转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。 关键词：小鼠; SV40 T; 转录激活域rtTA; PiggyBac 转座子; 正负向筛选 Construction and Integration Efficiency of PiggyBac Transposon Inducible Cell Immortalization Transgenic Vector ZHANG Ting-ting, XIN Ying, ZHANG Zhi-qiang, REN Chong-hua, YANG Han-jiang, WANG Ling, ZHANG Zhi-ying (Animal Genomics Laboratory, College of Animal Science and Technology, Northwest AF University, Yangling 712100, Shaanxi) Abstract: 【Objective 】 The objective of the study is to construct a Piggybac transposon-based transgenic vector for multiple gene expression and selection of removable marker gene. 【Method 】 To construct positive and negative selection marker gene cassette based on pXL-BacII, the two marker genes, neo (with SV40 promoter) and tk, were PCR amplified from the corresponding vectors and connected with IRES sequence by an overlap PCR approach. Then LoxP sequence was added into the expression cassette ends and resulted in PB-NIT. The transgenes, SV40 large T and mouse p 53 linked with T2A sequence, were firstly clo