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Nat Protoc. 2009;4(2):206-23. doi: 10.1038/nprot.2008.227.

Recombineering: a homologous recombination-based method of genetic engineering.

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1
Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. sharans@mail.nih.gov

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.

PMID:
19180090
PMCID:
PMC2790811
DOI:
10.1038/nprot.2008.227
[Indexed for MEDLINE]
Free PMC Article
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