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, oriR6Kγ, 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-bearingEscherichia 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 theloxP 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.
