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Recombineering: a homologous recombination-based method of genetic engineering

Nature Protocols volume 4, pages 206–223 (2009)Cite this article

Abstract

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about 1 week's time.

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Figure 1: Overview of bacteriophage λ recombination system used for recombineering.
Figure 2: A flowchart of recombineering procedures.
Figure 3: Insertion of a selectable marker by recombineering.
Figure 4: Insertion of a nonselectable DNA fragment by recombineering.
Figure 5: Subcloning DNA fragments from genomic DNA.
Figure 6: Two-step 'hit and fix' method to generate subtle mutations using ss short PCR product or oligonucleotides as targeting vector.
Figure 7: Schematic representation of λ phage constructs used for recombineering.

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Acknowledgements

We thank Nina Costantino and James Sawitzke for helpful comments. This research was supported by the Intramural Research Program of NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Authors and Affiliations

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

    Shyam K Sharan & Sergey G Kuznetsov

  2. SAIC-Frederick Inc., Gene Regulation and Chromosome Biology Laboratory, Basic Research Program, Frederick, 21702, Maryland, USA

    Lynn C Thomason

  3. Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, 21702, Maryland, USA

    Donald L Court

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Correspondence to Shyam K Sharan or Donald L Court.

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Sharan, S., Thomason, L., Kuznetsov, S. et al. Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4, 206–223 (2009). https://doi.org/10.1038/nprot.2008.227

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