Simple and efficient vectors for retrofitting BACs and PACs with mammalianneo(R) and EGFP marker genes

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs ) are widely used to investigate the functions of gents and genomes in mamm alian cells in vitro and in vivo. We have developed a series of vectors whi ch can simply and efficiently be retrofitted onto BACs or PACs. These vecto rs carry a neoR gene Tor selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick d etection of the DNA in cells. All the plasmids are retrofitted using the lo xP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitti ng of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be m onitored by the EGFP fluorescence. (C) 2001 Elsevier Science B.V. All right s reserved.