An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological Functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and Pi-derived artificial chromosomes (PACs] are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BAQ in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.