A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations

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Liu, Pentao, Jenkins, Nancy A., Copeland, Neal G.·2003·Genome Research

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Abstract

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the λ phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce lox P or FRT sites into the subcloned DNA. Unlike other methods that use short 45–55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive λ prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

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Authors: Liu, P., Jenkins, N. A., & Copeland, N. G.
Venue: Genome Research
Publisher: Cold Spring Harbor Laboratory
Published: 2003-03-01
Volume: 13
Issue: 3
Pages: 476-484
ISSN: 1088-9051 (ISSN-L)
1549-5469
DOI: 10.1101/gr.749203
OpenAlex: W2151214732
Cited by: 1,013
References: 0
Type: posted content
Language: en
Open access: bronze

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A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations

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