利用PCR产物快速构建兔HPRT 基因无启动子打靶载体

›› 2009, Vol. 29 ›› Issue (2): 86-92.

利用PCR产物快速构建兔HPRT 基因无启动子打靶载体

1.上海交通大学医学院实验动物科学部，上海，200025；2.新疆农业大学动物医学学院，乌鲁木齐，830052

收稿日期:2008-09-12 出版日期:2009-02-28 发布日期:2009-02-28

The Rapid Construction of Rabbit HPRT Promoterless Gene-targeting Vector with PCR Product

1. Department of Laboratory Animal Science, School of Medicine, Shanghai JiaoTong University Shanghai 200025, China 2. College of Animal Medicine of Xinjiang Agricultural University, Wulumuqi 830052, China

Received:2008-09-12 Online:2009-02-28 Published:2009-02-28

摘要/Abstract

摘要： 目的探讨构建兔HPRT基因打靶载体的方法，为下一步获得兔 HPRT基因敲除动物模型和转基因兔奠定基础。法首先在已经筛选到含有兔全长次黄嘌呤-鸟嘌呤磷酸核糖转移酶（Hypoxanthine guanine phosphoribosyl transferase，HPRT）基因BAC克隆（LBNL1-304M19）的基础上，利用Red重组系统，从此克隆上将一段12.5 kb无启动子的HPRT基因组片段克隆到pKS-质粒上，产生pKS-rHPRT质粒。后基于pKS-rHPRT和pIGCN21质粒，通过PCR的方法获得两个不同的带有50 bp HPRT 基因同源序列的IRES-eGFPCre-Frt/Neo/Frt同源重组片段，最终构建 HPRT 基因敲除打靶载体和无启动子基因敲入型打靶载体。果 PCR、限制性内切酶及DNA序列测定，表明载体构建成功。论成功快速地构建了HPRT 基因敲除打靶载体和无启动子基因敲入型打靶载体。时对利用同源重组技术敲除或插入DNA片段的效率进行了研究。

关键词: Red重组, PCR技术, 次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因（HPRT）, 打靶载体

Abstract: Objective To construct a knockout HPRT targeting vector and a promoterless knockin targeting vector,which can be used for making rabbit HPRT gene knockout models and transgenic rabbit in the future. Methods The rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HPRT gene fragment，which does not include promoter, is cloned into pKS-plasmid to form pKS-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, the PCR was used to obtain the IRES-eGFPCre-Frt/Neo/Frt fragments flanking with 50bp homologous arms for homologous recombination of rabbit HPRT gene on the basis of pKS-rHPRT and pIGCN21 plasmids. A knockout HPRT taigeting vector by replacing the last three exons with a IRES-eGFPCre-Frt/ Neo/Frt cassette and a promoterless knockin targeting vector by inserting a IRES-eGFPCre-Frt/Neo/Frt cassette in the 3’UTR of the HPRT gene were rapidly constructed. Results The identification of the vectors with PCR,enzyme restriction and sequence showed two vectors were constructed successfully. Conclusions We rapidly constructed a knockout HPRT taigeting vector and a promoterless knockin targeting vector, the efficiency of deleting or inserting DNA fragment by homologous recombination technology has also been studied.

Key words: Red recombination, PCR technology, HPRT gene, Targeting-vector

郭毅1，张传山2，谷瑞环1，姚刚2，陈学进1. 利用PCR产物快速构建兔HPRT 基因无启动子打靶载体[J]. , 2009, 29(2): 86-92.

GUO Yi1，ZHANG Chuan-shan2，GU Rui-huan1，YAO Gang2，CHEN Xue-jin1. The Rapid Construction of Rabbit HPRT Promoterless Gene-targeting Vector with PCR Product[J]. , 2009, 29(2): 86-92.