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Related Experiment Videos

A highly efficient recombineering-based method for generating conditional knockout mutations.

Pentao Liu1, Nancy A Jenkins, Neal G Copeland

  • 1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.

Genome Research
|March 6, 2003
PubMed
Summary
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A new recombineering method enables rapid generation of conditional mouse knockout (cko) mutations using lambda phage Red proteins and longer homology regions. This efficient technique facilitates functional genomic studies and the creation of gene editing tools.

Area of Science:

  • Molecular Biology
  • Genomics
  • Gene Engineering

Background:

  • Recombineering, a phage-based DNA modification technique in E. coli, offers alternatives to traditional restriction enzyme-based cloning.
  • Existing recombineering methods often rely on short homology regions, potentially limiting efficiency and scope.

Purpose of the Study:

  • To develop a novel recombineering-based method for efficiently generating conditional mouse knockout (cko) mutations.
  • To adapt recombineering for subcloning DNA from BACs and introducing specific recombination sites (loxP, FRT) for gene manipulation.

Main Methods:

  • Utilized lambda phage Red proteins for homologous recombination-mediated gap repair to subclone DNA from BACs into plasmids.
  • Employed Cre or Flpe recombinases to insert loxP or FRT sites into subcloned DNA.

Related Experiment Videos

  • Developed new E. coli strains with temperature-sensitive lambda prophage for recombination protein expression and arabinose-inducible promoters for recombinases.
  • Introduced novel Neo selection cassettes functional in both E. coli and mouse ES cells.

    • Main Results:

    • Demonstrated a recombineering method using significantly longer homology regions compared to previous approaches.
    • Successfully generated cko-targeting vectors in under two weeks, showcasing the method's speed and efficiency.
    • Validated the utility of new E. coli strains and selection cassettes for robust DNA engineering.

    Conclusions:

    • The described recombineering method provides a fast, efficient, and reliable approach for generating conditional mouse knockout (cko) mutations.
    • This technique simplifies the creation of targeting vectors, facilitating knock-in mutations, transgene construction, and the analysis of gene regulatory elements and functional domains.