利用Red重组系统快速构建基因打靶载体

中国生物工程杂志

2011, Vol. 31

Issue (10): 88-94

技术与方法

利用Red重组系统快速构建基因打靶载体

姜丽¹, 叶湘漓², 李大力¹

1. 华东师范大学生命科学学院生命医学研究所上海 200241;
2. 湖南师范大学医学院长沙 410013

Construction of Mouse Gene-targeting Vector Through Modified Recombineering Strategy

JIANG Li¹, YE Xiang-li², LI Da-li¹

1. Institute of Biomedical Sciences, East China Normal University, Shanghai 200241, China;
2. College of Medicine, Hunan Normal University, Changsha 410013, China

全文: PDF(574 KB) HTML

摘要：

基因敲除小鼠模型是在哺乳动物体内研究基因功能最可靠的方法之一。利用常规的分子克隆的方法构建基因打靶载体往往工作周期长,对于难度特别大的基因有时甚至无法完成打靶载体的构建。通过合理应用Red重组系统和低拷贝中间载体,利用50bp的同源重组序列直接从BAC载体中克隆了长片段的小鼠基因组序列;将得到的基因组序列再次通过重组和改造,构建了Gpr56等基因的完全敲除并带有报告基因的打靶载体,实现了打靶载体的快速构建。

关键词： 重组工程; 打靶载体构建; Red重组酶

Abstract:

Gene targeting is a well established technique which allows researchers to create virtually any desired modification in the genome of a living mouse. The first step to generate a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells. Since traditional construction strategy which mainly recruits PCR, restriction digestion and ligation is difficult to get longer DNA fragments for homologous recombination, A strategy using modified recombineering to generate gene targeting vectors was reported. Unlike other methods which use much longer PCR regions for construction, a method used short 50bp homologous regions which were synthesized in PCR primers as the homologous arms to clone more than 10 kb genomic DNA fragment from BAC which contains Gpr56 genomic DNA into a low copy vector through cap repair. Then the 2~5 exon region of Gpr56 was replaced by an ASCI-flanked cassette through a second recmobineering. After that, a DNA fragment which contains a Flp-FRT-based self-excision NEO cassette was then cloned into the vector with ligation through ASCI sites. After DNA sequencing of selected regions, it was confirmed that the Gpr56 targeting vector was what desired.

Key words: Recombineering Gene targeting Red recombinase

收稿日期: 2011-03-31 出版日期: 2011-10-25

ZTFLH:

Q789

基金资助:

国家自然科学基金(30800627);中央高校基本科研业务费专项资金(78210019)资助项目

通讯作者: 李大力 E-mail: dlli@bio.ecnu.edu.cn

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引用本文:

姜丽, 叶湘漓, 李大力. 利用Red重组系统快速构建基因打靶载体[J]. 中国生物工程杂志, 2011, 31(10): 88-94.

JIANG Li, YE Xiang-li, LI Da-li. Construction of Mouse Gene-targeting Vector Through Modified Recombineering Strategy. China Biotechnology, 2011, 31(10): 88-94.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I10/88

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