Figure 2.

A schematic representation of the two-step BAC modification system with pLD53.SC-AB vector. (A) The pLD53.SC-AB vector is based on the backbone of the plasmid pLD53 (Metcalf et al. 1996). A SalI-NotI fragment containing a SacB gene and a RecA gene was subcloned into the pLD53 vector. This shuttle vector carries a R6kγ origin, an ampicillin-resistant gene, and AscI and NotI sites as unique cloning sites for subcloning the recombination cassette, which contains a modification (insertion, deletion, or point mutation) flanked by two homology arms of ∼500 bp each. The two BAC modification steps are illustrated: (1) The co-integration of the shuttle vector pLD53.SC-AB into the BAC via homologous recombination through Box A; and (2) the resolution of the co-integrant involves a second homologous recombination event (either through two homology A or two homology B) to eliminate the pLD53 vector and other unnecessary sequences from the co-integrate. The resolved BACs were selected by growth on sucrose owing to the loss of SacB gene. (B) Southern blot analysis of a Huntington BAC clone modified by two-step BAC modification method. Co-integrates and resolved BAC candidates were first screened by PCR (data not shown). Positive clones were selected, and their DNA was digested with EcoRI, separated by electrophoresis on a 1% agarose gel, and transferred to nylon membrane. The blots were analyzed using the “A Box” as probes. Controls used were wild-type BAC DNA (B) and shuttle vector (V).