Construction of Gene-Targeting Vectors by Recombineering

  • Song-Choon Lee
  • Wei Wang
  • Pentao Liu
Part of the Methods in Molecular Biology book series (MIMB, volume 530)


Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by λ phage to manipulate DNA in Escherichia coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.

Key words

Conditional knockout recombineering vector gene targeting mouse E. coli 



This work is supported by The Wellcome Trust.


  1. 1.
    Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–6.PubMedCrossRefGoogle Scholar
  2. 2.
    Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 1981;78:7634–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Bradley A, Evans M, Kaufman MH, Robertson E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 1984;309:255–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987;51:503–12.PubMedCrossRefGoogle Scholar
  5. 5.
    Smithies O. Forty years with homologous recombination. Nat. Med. 2001;7:1083–6.PubMedCrossRefGoogle Scholar
  6. 6.
    Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 1988;336:348–52.PubMedCrossRefGoogle Scholar
  7. 7.
    van der Weyden L, Adams DJ, Bradley A. Tools for targeted manipulation of the mouse genome. Physiol. Genomics 2002;11:133–64.PubMedGoogle Scholar
  8. 8.
    Collins FS, Rossant J, Wurst W. A mouse for all reasons. Cell 2007;128:9–13.PubMedCrossRefGoogle Scholar
  9. 9.
    Bradley A, Hasty P, Davis A, Ramirez-Solis R. Modifying the mouse: design and desire. Biotechnology (NY) 1992;10:534–9.CrossRefGoogle Scholar
  10. 10.
    Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42.PubMedCrossRefGoogle Scholar
  11. 11.
    Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 1994;265:103–6.PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 1998;20:123–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Copeland NG, Jenkins NA, Court DL. Mouse genomic technologies recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2001;2:769–79.PubMedCrossRefGoogle Scholar
  14. 14.
    Court DL, Sawitzke JA, Thomason LC. Genetic engineering using homologous recombination. Annu. Rev. Genet. 2002;36:361–88.PubMedCrossRefGoogle Scholar
  15. 15.
    Sawitzke JA, Thomason LC, Costantino N, Bubunenko M, Datta S, Court DL. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 2007;421:171–99.PubMedCrossRefGoogle Scholar
  16. 16.
    Mythili E, Kumar KA, Muniyappa K. Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking. Gene 1996;182:81–7.PubMedCrossRefGoogle Scholar
  17. 17.
    Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 2000;97:5978–83.PubMedCrossRefGoogle Scholar
  18. 18.
    Cassuto E, Lash T, Sriprakash KS, Radding CM. Role of exonuclease and protein of phage lambda in genetic recombination. V. Recombination of lambda DNA in vitro. Proc. Natl. Acad. Sci. USA 1971;68:1639–43.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhang P, Li MZ, Elledge SJ. Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Nat. Genet. 2002;30:31–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 2000;97:6640–5.PubMedCrossRefGoogle Scholar
  21. 21.
    Murphy KC. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 1998;180:2063–71.PubMedGoogle Scholar
  22. 22.
    Lee EC, Yu D, Martinez de Velasco J, et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 2001;73:56–65.Google Scholar
  23. 23.
    Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 2003;13:476–84.PubMedCrossRefGoogle Scholar
  24. 24.
    Angrand PO, Daigle N, van der Hoeven F, Scholer HR, Stewart AF. Simplified generation of targeting constructs using ET recombination. Nucleic Acids Res. 1999;27:e16.PubMedCrossRefGoogle Scholar
  25. 25.
    Valenzuela DM, Murphy AJ, Frendewey D, et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotechnol. 2003;21:652–9.PubMedCrossRefGoogle Scholar
  26. 26.
    Cotta-de-Almeida V, Schonhoff S, Shibata T, Leiter A, Snapper SB. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria. Genome Res. 2003;13:2190–4.PubMedCrossRefGoogle Scholar
  27. 27.
    Chan W, Costantino N, Li R, et al. A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res. 2007;35:e64.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Song-Choon Lee
    • 1
  • Wei Wang
    • 1
  • Pentao Liu
    • 1
  1. 1.Wellcome Trust Sanger InstituteHinxtonUK

Personalised recommendations