利用细菌人工染色体同源重组对小鼠基因进行条件型敲除  

Conditional Knockout Construct for Mouse Gene by Bacterial Artificial Chromosome Homologous Recombination

作　　者：王立良[1] 王淼 王浩 张玉建 刘勇[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防与控制中心,北京100176 [2]University of Pennsylvania,Philadelphia [3]Baylor College of Medicine [4]MD Anderson Cancer Center,University of Texas

出　　处：《生物技术通讯》2010年第1期1-6,共6页Letters in Biotechnology

摘　　要：目的:探索通过细菌人工染色体(BAC)同源重组系统构建条件基因敲除载体的高效率方法,提高条件基因敲除小鼠(Flox小鼠)的构建效率。方法:利用作者自己构建的噬菌体重组酶系统,通过BAC同源重组进行条件型基因敲除载体构建工作。首先通过亚克隆构建了一系列载体含有同源臂的靶向质粒,线性化后,打靶片段经电穿孔法转入大肠杆菌内,与相应的BAC同源重组,再经过三步同源重组和一步位点特异性重组,构建小鼠条件型基因敲除载体。结果:高效率构建了小鼠基因的最终条件基因敲除载体。结论:通过BAC同源重组高效构建条件基因敲除载体,为条件基因敲除载体的构建提供了全新思路,并为FLox小鼠的建立,及相应基因在发育、生理、致病机制等方面的功能研究奠定了基础。Objective：To develop an effective way of making conditional knockout construct and Flox mice for gene function study,a novel strategy was explored by using bacterial artificial chromosome（BAC）-based homologous recombination.Methods：The conditional knockout vector was made by BAC recombination using our lambda phage recombinase system.A series of plasmids with homologous arms were constructed,and linearized target fragments were electroporated into E.coli and recombined with corresponding BAC.Three homologous recombination steps and one site-specific recombination step were used for conditional knockout vector construction.Results：A conditional knockout vector with targeted mouse gene flanked by two loxP sites was successfully constructed with high efficiency.Conclusion：A novel strategy based on BAC homologous recombination is developed to effectively generate conditional knockout construct.The method described here allows producing conditional knockout mice（Floxed mice）easily accomplished and facilitates gene function study in development or disease.

关 键 词：细菌人工染色体 同源重组 条件型基因敲除 打靶载体 Flox小鼠 

分 类 号：Q789[生物学—分子生物学]