Simple and highly efficient BAC recombineering using galK selection - PubMed (original) (raw)

Simple and highly efficient BAC recombineering using galK selection

Søren Warming et al. Nucleic Acids Res. 2005.

Abstract

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.

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Figures

Figure 1

Sequence analysis of the galactose operon in strains DY380 (A), SW101 (B) and SW102 (C). In SW102, the ORF of galK was deleted, leaving only 33 bp of galK behind to make sure that translation of galM is initiated properly. EcoRI: the restriction site used to clone the 5′ and 3′ homology arms flanking galK.

Figure 2

Overview of the galK selection scheme. The result of the first targeting event is the insertion of constitutively active galK into a defined position on the BAC by selection on minimal medium containing galactose and chloramphenicol to select for the maintenance of the BAC. The bacteria are now phenotypically Gal+. Next, the galK cassette is replaced by a dsDNA oligo, a PCR product, or a cloned dsDNA fragment carrying a desired mutation (indicated by a star) and flanked by the same homology arms used in the first selection step. This is achieved by negative selection using minimal medium containing 2-deoxy-galactose (DOG) with glycerol as the sole carbon source. The bacteria become phenotypically Gal−. H1 and H2, homology arms 1 and 2, respectively; cat, chloramphenicol acetyl transferase gene; ori2, BAC origin of replication; galK, E.coli galactokinase gene driven by a minimal promoter.

Figure 3

Introduction of a G12D mutation in the Nras gene. (A) SpeI restriction analysis of BAC miniprep DNA. First lane is the unmodified CITB-50J2 Nras BAC. Lanes 1–12 show digestion patterns of 12 clones counterselected for the substitution of galK with an oligo containing the G→A substitution for the second position of codon 12 of Nras. Clones 7 and 10 had internal deletions, indicating that DOG resistance was achieved by spontaneous deletion and not homologous recombination. These two clones were not analyzed further. (B) Sequence analysis of a PCR product spanning the modified region from clones 1–6, 8–9 and 11–12. All clones had the intended substitution (highlighted). However, clones 9 and 11 also had an internal basepair deletion indicated by a minus (highlighted). The Nras ATG and codon 12 are indicated (shadow).

Figure 4

BAC trimming using galK selection. (A) Illustration of the design of the deletion experiment. Homology arm 1 (H1) was held constant, and H2 was separated from H1 by either 50, 75 or 100 kb. (B) SpeI restriction analysis of BAC miniprep DNA from 12 clones showing deletions of 50, 75 and 100 kb, respectively, after the insertion of the galK selection cassette. The first lane is unmodified RP23-341F12 BAC DNA, which was included as a control. All tested clones had the intended deletion.

Figure 5

Insertion of a loxP511 site. (A) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via galK counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. (B) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a dsDNA oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. (C) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. (D) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site.

Figure 6

Same experiment as in Figure 5 with modifications as indicated at the top of each panel. (A) SpeI digest of 10 minipreps from a control experiment without heat-induction and without the loxP511 dsDNA oligo. (B) SpeI digest of 10 minipreps from a control experiment without heat-induction but with the loxP511 dsDNA oligo. (C) SpeI digest of 10 minipreps from a control experiment with heat-induction but without the loxP511 dsDNA oligo. (D) SpeI digest of 10 minipreps from an experiment with heat-induction and with the loxP511 dsDNA oligo (comparable with Figure 5B). Clones with the parental digestion pattern indicating DOG resistance due to homologous recombination (clones 33 and 35, circles) are only seen in (D). DOG resistance in all other clones likely occurred due to internal deletions of the BACs. (E) SpeI restriction analysis of BAC miniprep DNA from clones 33 and 35 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all the clones analyzed, confirming the correct insertion of loxP511 in clones 33 and 35 (compare with Figure 5C).

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Simple and highly efficient BAC recombineering using galK selection - PubMed (original) (raw)