title: Simple and highly efficient BAC recombineering using galK selection.
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.
authors.affiliation: Mouse Cancer Genetics Program, National Cancer Institute Frederick, MD 21702-1201, USA.
authors.forename: Søren
authors.initials: S
authors.lastname: Warming
authors.forename: Nina
authors.initials: N
authors.lastname: Costantino
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Jenkins
authors.forename: Neal G
authors.initials: NG
authors.lastname: Copeland
mesh_terms: D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005686:Galactokinase / Q000235:genetics*; D005690:Galactose / Q000378:metabolism; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009876:Operon; D017354:Point Mutation; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D029968:Escherichia coli Proteins; D005686:Galactokinase; D005690:Galactose
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
references.pmid: 10811905
references.citation: Nucleic Acids Res. 1999 Mar 15;27(6):1555-7
references.pmid: 10037821
references.citation: Genomics. 2001 Apr 1;73(1):56-65
references.pmid: 11352566
references.citation: Nat Rev Genet. 2001 Oct;2(10):769-79
references.pmid: 11584293
references.citation: Annu Rev Genet. 2002;36:361-88
references.pmid: 12429697
references.citation: Genome Res. 2002 Dec;12(12):1992-8
references.pmid: 12466304
references.citation: Genome Res. 2003 Mar;13(3):476-84
references.pmid: 12618378
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7207-12
references.pmid: 12771385
references.citation: Genomics. 2003 Jul;82(1):68-77
references.pmid: 12809677
references.citation: Genome Res. 2003 Sep;13(9):2190-4
references.pmid: 12915491
references.citation: Virology. 2004 Feb 20;319(2):185-9
references.pmid: 14980479
references.citation: J Bacteriol. 1975 Jan;121(1):259-66
references.pmid: 1090571
references.citation: Nucleic Acids Res. 1986 Mar 11;14(5):2287-300
references.pmid: 3457367
references.citation: Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645-9
references.pmid: 2162051
references.citation: Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8794-7
references.pmid: 1528894
references.citation: Cell. 1997 May 16;89(4):655-67
references.pmid: 9160756
references.citation: Nat Biotechnol. 1997 Sep;15(9):859-65
references.pmid: 9306400
references.citation: Nat Genet. 1998 Oct;20(2):123-8
references.pmid: 9771703
references.citation: Genesis. 2001 Jan;29(1):14-21
references.pmid: 11135458