Manipulating Genes and Gene Copy Number by Bacterial Artificial Chromosome Transfection

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the organization and activity of entire genes, replicons, and other large genomic loci. Protocols and parameters that influence the efficient transfection of these large DNA molecules into cells in culture were described here. By carefully optimizing the conditions for the formation of compact transfection complexes, BACs can be introduced into a variety of mammalian cells with reasonable efficiency. In addition, by cotransfection with a dihydrofolate reductase or hypoxanthine guanine phosphoribosyl transferase BAC, stable cell lines can be generated that carry 2–15 tandem chromosomal copies of the BAC of interest, thus providing a new avenue for studying gene dosage effects.