Modification and production of BAC transgenes.

Bacterial artificial chromosomes (BACs) are the vectors of choice for the construction of genomic DNA libraries and, as such, have proven instrumental in the generation of large-scale physical maps; positional cloning projects; and the sequencing of human, mouse, and a plethora of other genomes. A number of methods have recently been developed to modify BAC DNA (e.g., insertion, deletion, substitution), making BACs even more useful for functional genomic research. This unit describes two protocols for BAC modification in E. coli, one that allows for specific changes at a given DNA sequence and another that is more suited for rapid and nonspecific integration of foreign DNA (such as a reporter cassette) into a BAC insert. In addition, a simple and reliable method for preparing BAC DNA for pronuclear microinjection is also provided.