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

Abstract

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 P1-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 BACs 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.

Figure 1

Map of the retrofitting vector pRetroES and schematic illustration of the retrofitting procedure. (A) Map of pRetroES. Essentially, it has a GST-loxP-cre fusion gene driven by a tac promoter that encodes a fusion cre recombinase for promoting cre/loxP recombination. The conditional replication origin, ori^R6Kγ, will not function in the usual BAC host. The PGKneo is a selectable marker useful for embryonic stem cell transfection. (B) Schematic representation of the retrofitting procedure. In brief, a small volume of competent BAC-bearing Escherichia coli is prepared and transformed with pRetroES by electroporation. During the posttransformation incubation, the fusion cre recombinase will be expressed and will promote the recombination between the loxP sites on the retrofitting vector and the BAC vector, leading to the integration of pRetroES into the BAC at the loxP site. After integration, the tac promoter is separated from the fusion gene and no more cre recombinase will be produced. For detailed protocol, see Methods.

Figure 2

Representative results of retrofitted clones. (A) Schematic representation of possible recombinants from retrofitting pBACe3.6-generated BACs with pRetroES, showing primers used in colony PCR: F1 and F2 on the BAC vector and Retro-R (shown as R) on the retrofitting construct. (B) Colony PCR results showing the efficiency of the retrofitting and the frequency of integration at loxP and lox511 sites. Thirty-two chloramphenicol/ampicillin double-resistant colonies were PCR amplified with primers that identified integrations either at loxP site (F1 and R) or at lox511 site (F2 and R). As shown, 31 of the 32 clones have pRetroES integrated either at loxP (13/32) or at lox511 (19/32) site, and one of them, Clone 31, has a double integration. (C) Restriction digestion of five different retrofitted clones of the same BAC showing the fidelity of the retrofitting process. Five randomly picked cam/amp double-resistant colonies (numbered 1 through 5) were grown in liquid media and DNA prepared and digested with restriction enzymes AvrII, BamHI, and EcoRI. Electrophoresis was performed on 0.6% agarose at low voltage for about 24 h and the gel was stained with ethidium bromide.