Advertisement

Inducible Cre Mice

  • Susanne Feil
  • Nadejda Valtcheva
  • Robert Feil
Part of the Methods in Molecular Biology book series (MIMB, volume 530)

Abstract

The Cre/lox site-specific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. A major technical advance in terms of in vivo inducibility was the development of ligand-dependent Cre recombinases that can be activated by administration of tamoxifen to the animal. Here we describe how tamoxifen-dependent Cre recombinases, so-called CreER recombinases, work and how they can be used to generate time- and tissue-specific mouse mutants. The focus will be on the CreERT2 recombinase, which is currently the most successful CreER version. We will give an overview of available CreERT2 transgenic mouse lines and present protocols that detail the generation of experimental mice for inducible gene knockout studies, the induction of recombination by tamoxifen treatment, and the analysis of the quality and quantity of recombination by reporter gene and target gene studies. Most of the protocols can also be used as general guidelines for the generation and characterization of Cre/lox-mediated genome modifications in mice.

Key words

Transgenic mice inducible gene knockout somatic mutagenesis CreER recombinase tamoxifen in utero ROSA26 R26R X-Gal staining mouse models of human disease 

Notes

Acknowledgments

We thank the members of the Feil laboratory for critical discussion. Work in the authors’ laboratory was supported by grants from the VolkswagenStiftung and the Deutsche Forschungsgemeinschaft.

References

  1. 1.
    Branda CS, Dymecki SM. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 2004; 6:7–28.PubMedCrossRefGoogle Scholar
  2. 2.
    Feil R. Conditional somatic mutagenesis in the mouse using site-specific recombinases. Handb Exp Pharmacol. 2007:3–28.Google Scholar
  3. 3.
    Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P. Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA 1996; 93: 10887–90.PubMedCrossRefGoogle Scholar
  4. 4.
    Feil R, Wagner J, Metzger D, Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 1997; 237:752–7.PubMedCrossRefGoogle Scholar
  5. 5.
    Metzger D, Clifford J, Chiba H, Chambon P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci USA 1995; 92:6991–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang Y, Riesterer C, Ayrall AM, Sablitzky F, Littlewood TD, Reth M. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res 1996; 24:543–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Loonstra A, Vooijs M, Beverloo HB, et al. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA 2001; 98:9209–14.PubMedCrossRefGoogle Scholar
  8. 8.
    Schmidt EE, Taylor DS, Prigge JR, Barnett S, Capecchi MR. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA 2000; 97:13702–7.PubMedCrossRefGoogle Scholar
  9. 9.
    Indra AK, Warot X, Brocard J, et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 1999; 27:4324–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Kuhbandner S, Brummer S, Metzger D, Chambon P, Hofmann F, Feil R. Temporally controlled somatic mutagenesis in smooth muscle. Genesis 2000; 28:15–22.PubMedCrossRefGoogle Scholar
  11. 11.
    Soriano P. eneralized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999; 21:70–1.PubMedCrossRefGoogle Scholar
  12. 12.
    Gossen M, Bujard H. Studying gene function in eukaryotes by conditional gene inactivation. Annu Rev Genet 2002; 36:153–73.PubMedCrossRefGoogle Scholar
  13. 13.
    Sprengel R, Hasan MT. Tetracycline- controlled genetic switches. Handb Exp Pharmacol 2007:49–72.Google Scholar
  14. 14.
    Feil S, Hofmann F, Feil R. SM22alpha modulates vascular smooth muscle cell phenotype during atherogenesis. Circ Res 2004; 94:863–5.PubMedCrossRefGoogle Scholar
  15. 15.
    Lewandoski M. Analysis of mouse development with conditional mutagenesis. Handb Exp Pharmacol 2007:235–62.Google Scholar
  16. 16.
    Wolfsgruber W, Feil S, Brummer S, Kuppinger O, Hofmann F, Feil R. A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells. Proc Natl Acad Sci USA 2003; 100:13519–24.PubMedCrossRefGoogle Scholar
  17. 17.
    Lakso M, Pichel JG, Gorman JR, et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci USA 1996; 93:5860–5.PubMedCrossRefGoogle Scholar
  18. 18.
    Dupe V, Davenne M, Brocard J, et al. In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE). Development 1997; 124:399–410.PubMedGoogle Scholar
  19. 19.
    Furr BJ, Jordan VC. The pharmacology and clinical uses of tamoxifen. Pharmacol Ther 1984; 25:127–205.PubMedCrossRefGoogle Scholar
  20. 20.
    Brake RL, Simmons PJ, Begley CG. Cross-contamination with tamoxifen induces transgene expression in non-exposed inducible transgenic mice. Genet Mol Res 2004; 3:456–62.PubMedGoogle Scholar
  21. 21.
    Monvoisin A, Alva JA, Hofmann JJ, Zovein AC, Lane TF, Iruela-Arispe ML. VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium. Dev Dyn 2006; 235:3413–22.PubMedCrossRefGoogle Scholar
  22. 22.
    Kim JE, Nakashima K, de Crombrugghe B. Transgenic mice expressing a ligand-inducible cre recombinase in osteoblasts and odontoblasts: a new tool to examine physiology and disease of postnatal bone and tooth. Am J Pathol 2004; 165:1875–82.PubMedCrossRefGoogle Scholar
  23. 23.
    Chen M, Lichtler AC, Sheu TJ, et al. Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase. Genesis 2007; 45:44–50.PubMedCrossRefGoogle Scholar
  24. 24.
    Forde A, Constien R, Grone HJ, Hammerling G, Arnold B. Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 2002; 33:191–7.PubMedCrossRefGoogle Scholar
  25. 25.
    el Marjou F, Janssen KP, Chang BH, et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 2004; 39:186–93.PubMedCrossRefGoogle Scholar
  26. 26.
    Wen F, Cecena G, Munoz-Ritchie V, Fuchs E, Chambon P, Oshima RG. Expression of conditional cre recombinase in epithelial tissues of transgenic mice. Genesis 2003; 35:100–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Lantinga-van Leeuwen IS, Leonhard WN, van de Wal A, et al. Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cells. Genesis 2006; 44:225–32.PubMedCrossRefGoogle Scholar
  28. 28.
    Imai T, Jiang M, Chambon P, Metzger D. Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes. Proc Natl Acad Sci USA 2001; 98:224–8.PubMedCrossRefGoogle Scholar
  29. 29.
    Schuler M, Dierich A, Chambon P, Metzger D. Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse. Genesis 2004; 39:167–72.PubMedCrossRefGoogle Scholar
  30. 30.
    Hirrlinger PG, Scheller A, Braun C, Hirrlinger J, Kirchhoff F. Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2. Glia 2006; 54:11–20.PubMedCrossRefGoogle Scholar
  31. 31.
    Casper KB, Jones K, McCarthy KD. Characterization of astrocyte-specific conditional knockouts. Genesis 2007; 45:292–9.PubMedCrossRefGoogle Scholar
  32. 32.
    Mori T, Tanaka K, Buffo A, Wurst W, Kuhn R, Gotz M. Inducible gene deletion in astroglia and radial glia – a valuable tool for functional and lineage analysis. Glia 2006; 54:21–34.PubMedCrossRefGoogle Scholar
  33. 33.
    Imayoshi I, Ohtsuka T, Metzger D, Chambon P, Kageyama R. Temporal regulation of Cre recombinase activity in neural stem cells. Genesis 2006; 44:233–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Leone DP, Genoud S, Atanasoski S, et al. Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells.Mol Cell Neurosci 2003; 22:430–40.PubMedCrossRefGoogle Scholar
  35. 35.
    Schuler M, Ali F, Metzger E, Chambon P, Metzger D. Temporally controlled targeted somatic mutagenesis in skeletal muscles of the mouse. Genesis 2005; 41:165–70.PubMedCrossRefGoogle Scholar
  36. 36.
    Li M, Indra AK, Warot X, et al. Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis. Nature 2000; 407:633–6.PubMedCrossRefGoogle Scholar
  37. 37.
    Yajima I, Belloir E, Bourgeois Y, Kumasaka M, Delmas V, Larue L. Spatiotemporal gene control by the Cre-ERT2 system in melanocytes. Genesis 2006; 44:34–43.PubMedCrossRefGoogle Scholar
  38. 38.
    Bosenberg M, Muthusamy V, Curley DP, et al. Characterization of melanocyte-specific inducible Cre recombinase transgenic mice. Genesis 2006; 44:262–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Seibler J, Zevnik B, Kuter-Luks B, et al. Rapid generation of inducible mouse mutants. Nucl Acids Res 2003; 31:e12.PubMedCrossRefGoogle Scholar
  40. 40.
    Hameyer D, Loonstra A, Eshkind L, et al. Toxicity of ligand-dependent Cre-recombinases and generation of a conditional Cre-deleter mouse allowing mosaic recombination in peripheral tissues. Physiol Genomics 2007.Google Scholar
  41. 41.
    Santagati F, Minoux M, Ren SY, Rijli FM. Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis. Development 2005; 132:4927–36.PubMedCrossRefGoogle Scholar
  42. 42.
    Gruber M, Hu CJ, Johnson RS, Brown EJ, Keith B, Simon MC. Acute postnatal ablation of Hif-2alpha results in anemia. Proc Natl Acad Sci USA 2007; 104:2301–6.PubMedCrossRefGoogle Scholar
  43. 43.
    Ahn S, Joyner AL. Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 2004; 118:505–16.PubMedCrossRefGoogle Scholar
  44. 44.
    Zadelaar SM, Boesten LS, Pires NM, et al. Local cre-mediated gene recombination in vascular smooth muscle cells in mice. Transgenic Res 2006; 15:31–6.PubMedCrossRefGoogle Scholar
  45. 45.
    Stratis A, Pasparakis M, Markur D, et al. Localized inflammatory skin disease following inducible ablation of I kappa B kinase 2 in murine epidermis. J Invest Dermatol 2006; 126:614–20.PubMedCrossRefGoogle Scholar
  46. 46.
    Ouvrard-Pascaud A, Puttini S, Sainte-Marie Y, et al. Conditional gene expression in renal collecting duct epithelial cells: use of the inducible Cre-lox system. Am J Physiol Renal Physiol 2004; 286:F180–7.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Susanne Feil
    • 1
  • Nadejda Valtcheva
    • 1
  • Robert Feil
    • 1
  1. 1.Interfakultäres Institut für BiochemieUniversität TübingenGermany

Personalised recommendations