Advertisement

Molecular Biotechnology

, Volume 17, Issue 3, pp 269–275 | Cite as

Conditional gene knockout using cre recombinase

Protocol

Abstract

Cre recombinase has become an important instrument for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cre’s ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such conditional mutagenesis in mice depends both on the careful design of conditional alleles and on reliable detection of cre gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.

Index Entries

Cre site-specific recombination knockout FACS ES cells GFP STOP cassette fusion proteins transgenic mice loxP 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Gordon, J. W., Harold, G., and Leila, Y. (1993) Transgenic animal methodologies and their applications. Hum. Cell 6, 161–169.PubMedGoogle Scholar
  2. 2.
    Sauer, B. and Henderson, N. (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166–5170.PubMedCrossRefGoogle Scholar
  3. 3.
    Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994) Deletion of a polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265, 103–106.PubMedCrossRefGoogle Scholar
  4. 4.
    Lakso, M., Sauer, B., Mosinger, J., B., Lee, E. J., Manning, R. W., Yu. S.-H., Mulder, K. L., and Westphal, H. (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232–6236.PubMedCrossRefGoogle Scholar
  5. 5.
    Sauer, B. (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase, in “Guide to Techniques in Mouse Development”, Methods Enzymol. 225, 890–900.PubMedGoogle Scholar
  6. 6.
    Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y., Lee, E., Alt, F. W., and Westphal, H. (1996) Efficient in-vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865.PubMedCrossRefGoogle Scholar
  7. 7.
    Hoess, R. H. and Abremski, K. (1990) The Cre-lox recombination system, in, Nucleic Acids and Molecular Biology, vol. 4. (Eckstein, F. and Lilley, D. M. J., eds.), Springer-Verlag, Berlin, pp. 99–109.Google Scholar
  8. 8.
    Sauer, B., Whealy, M., Robbins, A., and Enquist, L. (1987) Site-specific insertion of DNA into a pseudorabies virus vector. Proc. Natl. Acad. Sci. USA 84, 9108–9112.PubMedCrossRefGoogle Scholar
  9. 9.
    Schwenk, F., Sauer, B., Kukoc, N., Hoess, R., Müller, W., Kocks, C., Kühn, R., and Rajewsky, K. (1997) Generation of Cre recombinase-specific monoclonal antibodies to characterize the pattern of Cre expression in cre-transgenic mouse strains. J. Immunol. Meth. 207, 203–212.CrossRefGoogle Scholar
  10. 10.
    Gagneten, S., Le, Y., Miller, J., and Sauer, B. (1997) Brief expression of a GFPcre fusion gene in embryonic stem cells allows rapid retrieval of sitespecific genomic deletions. Nucleic Acids Res. 25, 3326–3331.PubMedCrossRefGoogle Scholar
  11. 11.
    Le, Y., Miller, J., and Sauer, B. (1999) GFPcre fusion vectors with enhanced expression. Anal. Biochem. 270, 334–336.PubMedCrossRefGoogle Scholar
  12. 12.
    Gendron-Maguire, M., and Gridley, T. (1993) Identification of transgenic mice. Meth. Enzymol. 225, 794–799.PubMedGoogle Scholar
  13. 13.
    Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752.PubMedGoogle Scholar
  15. 15.
    Ramírez-Solis, R., Davis, A. C., and Bradley, A. (1993) Gene targeting in embryonic stem cells. Meth. Enzymol. 225, 855–878.PubMedCrossRefGoogle Scholar
  16. 16.
    Feiring, S., Kim, C. G., Epner, E. M., and Groudine, M. (1993) An “in-out” strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the β-globin locus control region. Proc. Natl. Acad. Sci. USA 90, 8469–8473.CrossRefGoogle Scholar
  17. 17.
    O’Gorman, S., Fox, D. T., and Wahl, G. M. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251, 1351–1355.PubMedCrossRefGoogle Scholar
  18. 18.
    Sauer, B. and Henderson, N. (1988) The cyclization of linear DNA in Escherichia coli by site-specific recombination. Gene 70, 331–341.PubMedCrossRefGoogle Scholar
  19. 19.
    Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y., Lee, E., Alt, F. W., and Westphal, H. (1996) Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865.PubMedCrossRefGoogle Scholar
  20. 20.
    Le, Y., Gagneten, S., Tombaccini, D., Bethke, B., and Sauer, B. (1999) Nuclear targeting determinants of the phage P1 Cre DNA recombinase. Nucleic Acids Res. 24, 4703–4709.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  1. 1.Developmental Biology ProgramOklahoma Medical Research FoundationOklahoma City

Personalised recommendations