Simple and highly efficient BAC recombineering using galK selection

By S. Warming, N. Costantino, D. Court, Nancy A. Jenkins, Neal G. Copeland. Nucleic Acids Research, 2005

1,349 citations recorded by the source corpus.

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (λ) 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 λ 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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2ba9a2242b84850f0f3275a3392dc7a2b53bc361

How researchers see this paper

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In Genetics

1. S. Warming
2. S. K. Sharan
3. Terence Hwa
4. M. Lamkanfi
5. S. Zipursky
6. D. Court
7. C. Tsui
8. Yi Chen

In Molecular Biology

1. M. Lamkanfi
2. V. Dixit
3. S. Warming
4. S. K. Sharan
5. Terence Hwa
6. José M. Alonso
7. S. Zipursky
8. M. Krüger

Citations in the corpus

• Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence (2011, methodology): “Improvements in cloning methods used for constructing transgene”
• Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster. (2009, methodology): “region with a galK cassette (45) that was amplified by the prim-”
• Immunogenicity of induced pluripotent stem cells (2011, neutral): “E. coli strain SW106 was transformed with BAC DNA spanning human ATM, p53, or HPRT locus (Warming et al., 2005).”
• TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. (2008, methodology): “Whole-gene translational fusions for TAA1 and TAR1 with GFP were made using bacterial recombineering approach (Warming et al., 2005).”
• Recombineering: A Homologous Recombination-Based Method of Genetic Engineering (2009, background): “gene linked with a drug marker, such as cat or kan , the galK gen”
• Growth-rate dependent global effects on gene expression in bacteria (2009, methodology): “with a constitutive promoter was amplified from pgalk (Warming et al., 2005) using”
• Cell-type Specific Labeling of Synapses in vivo through Synaptic Tagging with Recombination (STaR) (2014, methodology): “We modified the endogenous brp sequence in a BAC by inserting a V5 tag into the brp coding region downstream from transcriptional and translational stop sequences flanked by FRT recombination sites (Golic and Lindquist, 1989; Southern et al., 1991; Venken et al., 2006; Warming et al., 2005).”
• Non-canonical inflammasome activation targets caspase-11 (2011, methodology): “Correctly targeted BAC DNA was transformed into Flp-expressing SW105 cell”