Abstract
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. genesis 38:39–50, 2004. © 2003 Wiley‐Liss, Inc.