Modification of bacterial artificial chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice.
Modification of bacterial artificial chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice.
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DOI：
10.1002/0471142301.ns0521s31
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发表时间：
2005-05-01
影响因子：
--
通讯作者：
Yang, X William
中科院分区：
文献类型：
--
作者：
Gong, Shiaoching;Yang, X William
BAC转基因是研究小鼠体内基因表达和基因功能的有力工具。本单元提供了在大肠杆菌中通过同源重组对BACs进行修饰（即标记基因插入、删除或点突变）的详细方案。该方法利用穿梭载体，允许大肠杆菌RecA基因的瞬时表达，以支持在BAC宿主细菌中的同源重组。此外，还提供了两种纯化BAC DNA用于显微注射以产生转基因小鼠的方法。由于BAC DNA容易降解，可能在转基因小鼠中引入位置效应，因此给出了两种纯化完整BAC DNA的方法，用于随后的显微注射。
BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.