Skip to main content

Targeted Somatic Mutagenesis in the Mouse Epidermis

  • Protocol
Epidermal Cells

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 289))

  • 1336 Accesses

Abstract

The efficient introduction of somatic mutations in a given gene at a given time and in specific cell types of the skin will greatly facilitate the studies of a number of genes expressed in the skin and the production of animal models for skin diseases. We describe here strategies and techniques to create spatiotemporally controlled somatic mutations of target genes in the skin using a conditional Cre/LoxP system. They are based on cell-specific expression of the chimeric Cre recombinase Cre-ERT2, whose activity is induced by antiestrogens such as Tamoxifen (Tam), and which is obtained by fusing the Cre recombinase with a mutated ligand binding domain of the human estrogen receptor ERα. As an example, we present ablation of the retinoid receptor RXRα in epidermal basal keratinocytes of adult mice.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Metzger, D., and Chambon, P. (2001) Site-and time-specific gene targeting in the mouse. Methods 24, 71–80.

    Article  PubMed  CAS  Google Scholar 

  2. Jonkers, J. and Berns, A. (2002) Conditional mouse models of sporadic cancer. Nat. Rev. Cancer 2, 251–265.

    Article  PubMed  CAS  Google Scholar 

  3. Rajewsky, K., Gu, H., Kuhn, R., Betz, U. A., Muller, W., Roes, J., et al. (1996) Conditional gene targeting. J. Clin. Invest. 98, 600–603.

    Article  PubMed  CAS  Google Scholar 

  4. Lewandoski, M. (2001) Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743–755.

    Article  PubMed  CAS  Google Scholar 

  5. Zhu, Z., Zheng, T., Lee, C. G., Homer, R. J., and Elias, J. A. (2002) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin Cell Dev. Biol. 13, 121–128.

    Article  PubMed  CAS  Google Scholar 

  6. Metzger, D. and Feil, R. (1999) Engineering the mouse genome by site-specific recombination. Curr. Opin. Biotechnol. 10, 470–476.

    Article  PubMed  CAS  Google Scholar 

  7. Garcia, E. L. and Mills, A. A. (2002) Getting around lethality with inducible Cre-mediated excision. Semin. Cell Dev. Biol. 13, 151–158.

    Article  PubMed  CAS  Google Scholar 

  8. Metzger, D., Indra, A. K., Li, M., Chapellier, B., Calleja, C, Ghyselinck, N., et al. (2003) Targeted conditional somatic mutagenesis in the mouse: temporally-controlled knock out of retinoid receptors in epidermal keratinocytes. Meth. Enzymol. 364, 379–408.

    PubMed  CAS  Google Scholar 

  9. Fuchs, E. (1997) Keith R. Porter Lecture, 1996. Of mice and men: genetic disorders of the cytoskeleton. Mol. Biol. Cell 8, 189–203.

    PubMed  CAS  Google Scholar 

  10. Hardy, M. H. (1992) The secret life of the hair follicle. Trends Genet. 8, 55–61.

    PubMed  CAS  Google Scholar 

  11. Paus, R. and Cotsarelis, G. (1999) The biology of hair follicles. N. Engl. J. Med. 341, 491–497.

    Article  PubMed  CAS  Google Scholar 

  12. Mascrez, B., Mark, M., Dierich, A., Ghyselinck, N. B., Kastner, P., and Chambon, P. (1998) The RXRalpha ligand-dependent activation function 2 (AF-2) is important for mouse development. Development 125, 4691–4707.

    PubMed  CAS  Google Scholar 

  13. Soriano, P. (1999) Generalized LacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71.

    Article  PubMed  CAS  Google Scholar 

  14. Li, M., Chiba, H., Warot, X., Messaddeq, N., Gérard, C, Chambon, P., et al. (2001) RXRalpha ablation in skin keratinocytes results in alopecia and epidermal alterations. Development 128, 675–688.

    PubMed  CAS  Google Scholar 

  15. Vassar, R., Rosenberg, M., Ross, S., Tyner, A., and Fuchs, E. (1989) Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc. Natl. Acad. Sci. USA 86, 1563–1567.

    Article  PubMed  CAS  Google Scholar 

  16. Li, M., Indra, A. K., Warot, X., Brocard, J., Messaddeq, N., Kato, S., et al. (2000) Skin abnormalities generated by temporally controlled RXRαlpha mutations in mouse epidermis. Nature 407, 633–636.

    Article  PubMed  CAS  Google Scholar 

  17. Sambrook, J. and Russel, D. W. (2001) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  18. Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. (2003) Manipulating the Mouse Embryo. A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  19. Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. (1996) Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. USA 93, 10,887–10,890.

    Article  PubMed  CAS  Google Scholar 

  20. Brocard, J., Warot, X., Wendling, O., Messaddeq, N., Vonesch, J. L., Chambon, P., et al. (1997) Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc. Natl. Acad. Sci. USA 94, 14,559–14,563.

    Article  PubMed  CAS  Google Scholar 

  21. Weber, P., Metzger, D., and Chambon, P. (2001). Temporally controlled targeted somatic mutagenesis in the mouse brain. Eur. J. Neurosci. 14, 1777–1783.

    Article  PubMed  CAS  Google Scholar 

  22. Indra, A. K., Warot, X., Brocard, J., Bornert, J. M., Xiao, J. H., Chambon, P., et al. (1999) 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. 27, 4324–4327.

    Article  PubMed  CAS  Google Scholar 

  23. Lobe, C. G, Koop, K. E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. (1999). Z/AP, a double reporter for cre-mediated recombination. Dev. Biol. 208, 281–292.

    Article  PubMed  CAS  Google Scholar 

  24. Novak, A., Guo, C, Yang, W., Nagy, A., and Lobe, C. G. (2000) Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147–155.

    Article  PubMed  CAS  Google Scholar 

  25. Akagi, K., Sandig, V., Vooijs, M., Van der Valk, M., Giovannini, M., Strauss, M.,et al. (1997) Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 25, 1766–1773.

    Article  PubMed  CAS  Google Scholar 

  26. Weber, P., Schuler, M., Gérard, C, Mark, M., Metzger, D., and Chambon, P. (2003) Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis. Biol. Reprod. 68, 553–559.

    Article  PubMed  CAS  Google Scholar 

  27. Imai, T., Jiang, M., Chambon, P., and Metzger, D. (2001) 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 98, 224–228.

    Article  PubMed  CAS  Google Scholar 

  28. Imai, T., Jiang, M., Kastner, P., Chambon, P., and Metzger, D. (2001). Selective ablation of retinoid X receptor alpha in hepatocytes impairs their lifespan and regenerative capacity. Proc. Natl. Acad. Sci. USA 98, 4581–4586.

    Article  PubMed  CAS  Google Scholar 

  29. Yang, X. W., Model, P., and Heintz, N. (1997) Homologous recombination based modification in Escherichia coli and germline transmission in trangenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865.

    Article  PubMed  CAS  Google Scholar 

  30. Bell, A. C, West, A. G., and Felsenfeld, G. (2001) Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science 291, 447–450.

    Article  PubMed  CAS  Google Scholar 

  31. Giraldo, P., and Montoliu, L. (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 10, 83–103.

    Article  PubMed  CAS  Google Scholar 

  32. Bailleul, B., Surani, M. A., White, S., Barton, S. C, Brown, K., Blessing, M., et al. (1990) Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell 62, 697–708.

    Article  PubMed  CAS  Google Scholar 

  33. Greenhalgh, D. A., Rothnagel, J. A., Quintanilla, M. I., Orengo, C. C, Gagne, T. A., Bundman, D. S., et al. (1993) Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene. Mol. Carcinog. 7, 99–110.

    Article  PubMed  CAS  Google Scholar 

  34. Kishimoto, J., Ehama, R., Wu, L., Jiang, S., Jiang, N., and Burgeson, R. E. (1999) Selective activation of the versican promoter by epithelial-mesenchymal interactions during hair follicle develoment. Proc. Natl. Acad. Sci. USA 96, 7336–7341.

    Article  PubMed  CAS  Google Scholar 

  35. Porter, S. D., and Meyer, C. J. (1994) A distal tyrosinase upstream element stimulates gene expression in neural-crest-derived melanocytes of transgenic mice: position-independent and mosaic expression. Development 120, 2103–2111.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Metzger, D., Li, M., Chambon, P. (2005). Targeted Somatic Mutagenesis in the Mouse Epidermis. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology™, vol 289. Humana Press. https://doi.org/10.1385/1-59259-830-7:329

Download citation

  • DOI: https://doi.org/10.1385/1-59259-830-7:329

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-267-4

  • Online ISBN: 978-1-59259-830-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics