Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome
Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome
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DOI：
10.1038/nbt0997-859
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发表时间：
1997-09-01
影响因子：
46.9
通讯作者：
Heintz, N
中科院分区：
文献类型：
--
作者：
Yang, XDW;Model, P;Heintz, N
基于大肠杆菌的人工染色体已成为各种基因组计划中物理作图和测序的重要工具。然而，缺乏一种通用的方法来修饰这些大型细菌克隆，限制了它们在功能研究中的应用。我们建立了一种简单的方法，通过同源重组直接修饰重组缺陷大肠杆菌宿主菌株的细菌人工染色体，用于体内研究。将IRES-LacZ标记基因导入含有鼠锌指基因RU49的131 kb BAC中。在修饰的bac中未检测到重排或缺失。此外，通过原核注射获得了转基因小鼠，并获得了完整BAC的种系传播。在脑内观察到lacZ基因的正确表达，这是传统转基因结构所不能达到的。
Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAC containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAG, and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.