General method, for the modification of different BAC types and the rapid generation of BAC transgenic mice
General method, for the modification of different BAC types and the rapid generation of BAC transgenic mice
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DOI：
10.1002/gene.10249
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发表时间：
2004-01-01
期刊：
影响因子：
1.5
通讯作者：
Eberl, G
中科院分区：
文献类型：
--
作者：
Sparwasser, T;Gong, SC;Eberl, G
大多数基因组计划依赖于细菌人工染色体（BAC）的测序，其包含100-300 kb的基因组DNA。结果，几千个BAC克隆现在被映射到人类和小鼠的基因组中。因此，有可能通过计算机识别携带特定基因的BAC克隆并将其商业化。考虑到BAC的大尺寸，基因的大多数（如果不是全部的话）调节序列存在，并且可以用于指导异源基因在体外细胞培养物中和在BAC转基因小鼠体内的忠实和组织特异性表达。我们在这里描述了一个优化和全面的协议，以选择，修改和纯化BAC，以产生BAC转基因小鼠。重要的是，该方案包括在2天内产生修饰BAC所需的复合质粒盒的方法，以及有效地修饰选自两种主要BAC文库的不同类型的BAC的方法。总而言之，我们的方法结合使用基因组数据库分析、重叠PCR克隆和细菌中的BAC重组，可以快速可靠地产生“假敲入”小鼠。(C)2003 Wiley-Liss，Inc.
Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. (C) 2003 Wiley-Liss, Inc.