重组工程系统研究进展  

Advances in recombineering system

作　　者：于梅[1] 周建光[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2004年第6期617-619,共3页Letters in Biotechnology

基　　金：全军"十五"医药卫生科研基金(01MA089)

摘　　要：大肠杆菌的同源重组依靠内源性的RecA蛋白。RecBCD降解双链线性DNA分子,因此必须构建环状质粒打靶载体才能完成体内重组,操作过程繁琐,所需同源臂长。最近建立起的依赖于Rac噬菌体的ET重组系统和基于λ噬菌体的Red重组系统,可有效利用线性DNA片段作为打靶分子,对大肠杆菌染色体DNA和BAC、PAC载体中包含的真核细胞基因组DNA进行基因敲除、敲入、替换、单碱基突变及体内基因克隆等修饰。这2种系统重组效率高,用PCR方法便可合成双链线性DNA打靶分子,不需要限制酶和连接酶,操作过程简单、精确、快速、经济,大大缩短了构建打靶载体的时间,成为功能基因组研究的有力工具。In Escherichia coli most recombinations depend on intracellular RecA protein. RecBCD degrades double-stranded DNA, so only circular dsDNA with long homology as targeting cassettes can carry out recombination in vivo. Recently, highly efficient phage-based E.coli homologous recombination systems including Rac-encoded RecET system and λ-encoded Red system have been developed. These phage systems can catalyze efficient recombination with very short homology segments. Mutation such as insertion, deletion, substitution, single-base changes and in vivo cloning by gap repair can be introduced into E.coli chromosome DNA or eukaryotic genomics DNA cloned in BACs or PACs. Importantly they need no restriction enzymes and DNA ligase, eliminate many of the time-consuming in vitro steps of construction. Recombineering offers a powerful new tool for functional genomics.

关 键 词：体内 靶分子 ET 大肠杆菌 打靶载体 BAC 内源性 重组工程 双链 DNA分子 

分 类 号：Q78[生物学—分子生物学] R373[医药卫生—病原生物学]