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

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

1,117 citations recorded by the source corpus.

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 lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP 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 lambda 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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/efe3fdf40bb62d029e0c608c70ae21ce9cb0902c

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Citations in the corpus

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. (2012, neutral): “Recently, Prom1 has been found to be phosphorylated by Src (54), and knockdown of Prom1 or treatment with a Src inhibitor was shown to suppress the of pre-Mir122a was cloned into PL253 in the bacterial strain EL350 by a recombineering-based method (19).”
Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. (2008, positive): “using appropriate restriction enzymes (miR-17� 92 and miR-106a� 363 )o r by ‘‘recombineering’’ ( Liu et al., 2003 )( miR-106b� 25).”
Recombineering: A Homologous Recombination-Based Method of Genetic Engineering (2009, positive): “coli , and these modified constructs can then be used to generate knockout and knock-in mouse models using embryonic stem cell technolog”
A global double‐fluorescent Cre reporter mouse (2007, methodology): “Following verification of construct function in vitro, an FRT-flanked Neo cassette (selectable marker) was subcloned distal to the mG-pA in pCA-mT/mG via EcoRI and AscI restriction sites added to the 50 and 30 ends of pL451 (NCI) (Liu et al., 2003).”
Endothelial and perivascular cells maintain haematopoietic stem cells (2012, methodology): “Methods Summary Targeting vectors for making Scf gfp and Scf fl mice were constructed by recombineerin”
The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals (2010, methodology): “Generation of Yap Conditional Knock-Out Mice A targeting vector containing the first two exons of the Yap gene was generated by recombineering as described previously (Liu et al., 2003).”
Simple and highly efficient BAC recombineering using galK selection (2005, methodology): “The resulting strains, SW105 (Flp) and SW106 (Cre) (Table 1), were then tested to confirm that they still contained inducible Flp and Cre genes by transforming arabinose-induced and electrocompetent cells with plasmids containing a neo gene flanked by Frt or loxP sites, PL451 and PL452 (4), respecti”
Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon (2009, neutral): “Finally, PGKneo cassette was excised from PL452 (ref”
Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth (2012, methodology): “Retrieval of the targeting region (covering 753-12,812; numbering relative to indicated BamHI site) was performed according to Liu et al.(34) using C57BL/6 bacterial artificial chromosome RP23-283M8 (BACPAC Resources) as template in DY380 cells.”