Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication

Abstract

Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

Figure 1

(A) Efficient modification of a Zipro1 BAC by using R6kγ RecA-based shuttle vector. A 500-bp fragment corresponding to the last exon of the Zipro 1 gene (Yang et al. 1997) and an IRESEGFP marker gene were cloned into pLD53-RecA vector. This shuttle vector was then electroporated into Zipro1 containing BAC169 cells (Yang et al. 1997). Co-integrates were formed via homologous recombination and selected by chloramphenicol and ampicillin. (B) Southern blot analysis of the co-integrate BAC clones. Co-integrates BAC DNA were digested with SpeI and separated by electrophoresis on a 1% agarose gel. The DNA was transferred to nylon membrane, and the blot was analyzed by using the “A Box” as a probe: co-integrate BAC clones (lanes 1–16), wild-type BAC (lane B), and shuttle vector (lane V).

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).

Figure 3

A schematic representation of the two-step BAC modification system with pLD53.SC1 vector. This vector is based on the backbone of the plasmid pLD53 (Metcalf et al. 1996). A SalI-Not1 fragment containing a SacB gene, a RecA gene, two IRESEGFP genes, and multiple cloning sites was subcloned into a mutated pLD53. This shuttle vector carries a R6kγ origin, an ampicillin-resistant gene, and AscI and SmaI as unique cloning sites for subcloning of Box A. The two BAC modification steps are illustrated: (1) The co-integration of the shuttle vector pLD53.SC1 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 IRESEGFP) to eliminate the pLD53 vector and other unnecessary sequences from the co-integrate. The resolved BACs are selected by growth on sucrose owing to the loss of SacB gene.

Figure 4

Southern blot analysis of clones modified by the pLD53.SC1 shuttle vector with the built-in resolution strategy. Co-integrates and resolved BAC DNA were digested with SpeI, 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 (lane B) and shuttle vector (lane V).