Skip to main content
SpringerLink
Log in
Book cover

The Nuclear Receptor Superfamily pp 237–261Cite as

Tissue-Selective Knockouts of Steroid Receptors: A Novel Paradigm in the Study of Steroid Action

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 505))

Abstract

The use of tissue-selective rather than ubiquitous knockouts of steroid receptors allows a more refined study of the mechanism of steroid action in defined target tissues and circumvents problems such as early lethality or major developmental defects precluding studies in affected organs. In this chapter, we describe the main steps involved in the development of tissue-selective steroid receptor knockouts by Cre/loxP technology. Problems in the development of a mouse strain with a floxed receptor allele, the selection of a suitable Cre expressing mouse strain, the generation of cell-selective knockouts by crossbreeding of the mentioned mouse strains, and the control of appropriate receptor inactivation are discussed taking the generation of mice with a Sertoli cell-selective ablation of the androgen receptor as an example.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Vogel, G. (2007) Nobel prizes—A knockout award in medicine. Science,318, 178–179.

    Article  CAS  PubMed  Google Scholar 

  2. Schmid, W., Cole,T.J., Blendy,J.A., and Schutz, G. (1995) Molecular genetic analysis of glucocorticoid signalling in development. Journal of Steroid Biochemistry and Molecular Biology, 53, 33–35.

    Article  CAS  PubMed  Google Scholar 

  3. De Gendt, K., Swinnen, J.V., Saunders, P.T.K., Schoonjans, L., Dewerchin, M., Devos, A., Tan, K., Atanassova, N., Claes-sens, F., Lecureuil, C., Heyns, W., Car-meliet, P., Guillou, F., Sharpe, R.M., and Verhoeven, G. (2004) A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis.Proceedings of the National Academy of Sciences USA, 101, 1327–1332.

    Article  Google Scholar 

  4. Kellendonk, C., Tronche, F., Casanova, E., Anlag,K., Opherk,C., and Schutz,G. (1999) Inducible site-specific recombination in the brain. Journal of Molecular Biology, 285, 175–182.

    Article  CAS  PubMed  Google Scholar 

  5. Sauer, B. (1998) Inducible gene targeting in mice using the Cre/lox system. Methods-A Companion to Methods in Enzymology, 14, 381–392.

    Article  CAS  Google Scholar 

  6. Nagy, A. (2000) Cre recombinase: The universal reagent for genome tailoring. Genesis, 26, 99–109.

    Article  CAS  PubMed  Google Scholar 

  7. De Gendt, K., Atanassova, N., Tan, K.A.L., de Franca, L.R., Parreira, G.G., Mckinnell, C., Sharpe, R.M., Saunders, P.T.K., Mason, J.I., Hartung, S., Ivell, R., Denolet, E., and Verhoeven, G. (2005) Development and function of the adult generation of Ley-dig cells in mice with Sertoli cell-selective or total ablation of the androgen receptor. Endocrinology, 146, 4117–4126.

    Article  PubMed  Google Scholar 

  8. Denolet, E., De Gendt, K., Allemeersch, J., Engelen, K., Marchal, K., Van Hummelen, P., Tan, K.A.L., Sharpe, R.M., Saunders, P.T.K., Swinnen, J.V., and Verhoeven, G. (2006) The effect of a Sertoli cell-selective knockout of the androgen receptor on tes-ticular gene expression in prepubertal mice. Molecular Endocrinology, 20, 321–334.

    Article  CAS  PubMed  Google Scholar 

  9. Tan, K.A.L., De Gendt, K., Atanassova, N., Walker, M., Sharpe, R.M., Saunders, P.T.K., Denolet, E., and Verhoeven,G. (2005) The role of androgens in Sertoli cell proliferation and functional maturation: Studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology, 146, 2674–2683.

    Article  CAS  PubMed  Google Scholar 

  10. Sauer, B. and Henderson, N. (1988) Site-specific DNA recombination in mammalian-cells by the Cre recombinase of bacteriophage-P1. Proc.Natl.Acad. Sci.U.S.A., 85, 5166–5170.

    Article  CAS  PubMed  Google Scholar 

  11. Hellwinkel, O.J.C., Bull, K., Holter-hus, P.M., Homburg, N., Struve, D., and Hiort,O. (1999) Complete androgen insensitivity caused by a splice donor site mutation in intron 2 of the human andro-gen receptor gene resulting in an exon 2-lacking transcript with premature stop-codon and reduced expression. Journal of Steroid Biochemistry and Molecular Biology, 68, 1–9.

    Article  CAS  PubMed  Google Scholar 

  12. Yeh, S.Y., Tsai, M.Y., Xu, Q.Q., Mu, X.M., Lardy, H., Huang, K.E., Lin, H., Yeh, S.D., Altuwaijri,S., Zhou, X.C., Xing, L.P., Boyce, B.F., Hung, M.C., Zhang,S., Gan, L., and Chang, C.S. (2002) Generation and characterization of androgen receptor knockout (ARKO) mice: An in vivo model for the study of androgen functions in selective tissues. Proceedings of the National Academy of Sciences USA, 99, 13498–13503.

    Article  CAS  Google Scholar 

  13. De Gendt, K., Atanassova, N., Tan, K.A.L., de Franca, L.R., Parreira, G.G., Mckinnell, C., Sharpe, R.M., Saunders, P.T.K., Mason, J.I., Hartung, S., Ivell, R., Denolet, E., and Verhoeven, G. (2005) Development and function of the adult generation of Ley-dig cells in mice with Sertoli cell-selective or total ablation of the androgen receptor. Endocrinology, 146, 4117–4126.

    Article  PubMed  Google Scholar 

  14. Tybulewicz, V.L.J., Crawford, C.E., Jackson, P.K., Bronson, R.T., and Mulligan, R.C. (1991) Neonatal lethality and lym-phopenia in mice with a homozygous disruption of the C-Abl protooncogene. Cell, 65, 1153–1163.

    Article  CAS  PubMed  Google Scholar 

  15. Nagy, A., Rossant, J., Nagy, R., Abramownew-erly, W., and Roder, J.C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem-cells. Proceedings of the National Academy of Sciences USA, 90, 8424–8428.

    Article  CAS  Google Scholar 

  16. O'Gorman, S., Dagenais, N.A., Qian, M., and Marchuk, Y. (1997) Protamine-Cre recombinase transgenes efficiently recom-bine target sequences in the male germ line of mice, but not in embryonic stem cells. Proceedings of the National Academy of Sciences USA, 94, 14602–14607.

    Article  Google Scholar 

  17. Schoonjans, L., Kreemers, V., Danloy, S., Moreadith, R.W., Laroche, Y., and Collen,D. (2003) Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains. Stem Cells, 21, 90–97.

    Article  PubMed  Google Scholar 

  18. Lecureuil, C., Fontaine, I., Crepieux, P., and Guillou, F. (2002) Sertoli and granu-losa cell-specific Cre recombinase activity in transgenic mice. Genesis, 33, 114–118.

    Article  CAS  PubMed  Google Scholar 

  19. Bremner, W.J., Millar, M.R., Sharpe, R.M., and Saunders, P.T.K. (1994) Immuno-histochemical Localization of Androgen Receptors in the Rat Testis–Evidence for Stage-Dependent Expression and Regulation by Androgens. Endocrinology, 135, 1227–1234.

    Article  CAS  PubMed  Google Scholar 

  20. Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genetics, 21, 70–71.

    Article  CAS  PubMed  Google Scholar 

  21. Lindsey, J.S. and Wilkinson, M.F. (1996) Pem: A testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. Developmental Biology, 179, 471–484.

    Article  CAS  PubMed  Google Scholar 

  22. Schwenk, F., Baron, U., and Rajewsky, K. (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Research, 23, 5080–5081.

    Article  CAS  PubMed  Google Scholar 

  23. Lewandoski, M. and Martin, G.R. (1997) Cre-mediated chromosome loss in mice. Nature Genetics, 17, 223–225.

    Article  CAS  PubMed  Google Scholar 

  24. Lallemand, Y., Luria,V., Haffner-Krausz, R., and Lonai, P. (1998) Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase. Transgenic Research, 7, 105–112.

    Article  CAS  PubMed  Google Scholar 

  25. Tsai, M.Y., Yeh, S.D., Wang, R.S., Yeh, S., Zhang, C., Lin, H.Y., Tzeng, C.R., and Chang, C. (2006) Differential effects of spermatogenesis and fertility in mice lacking androgen receptor in individual testis cells. Proceedings of the National Academy of Sciences USA, 103, 18975–18980.

    Article  CAS  Google Scholar 

  26. Zhang, C.X., Yeh,S.Y., Chen, Y.T., Wu, C.C., Chuang, K.H., Lin, H.Y., Wang, R.S., Chang,Y.J., Mendis-Handagama,C., Hu, L.Q., Lardy, H., and Chang, C.S. (2006) Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells. Proceedings of the National Academy of Sciences USA, 103, 17718–17723.

    Article  CAS  Google Scholar 

  27. Simanainen, U., Allan, C.M., Lim, P., McPherson, S., Jimenez, M., Zajac, J.D., Davey, R.A., and Handelsman, D.J. (2007) Disruption of prostate epithelial androgen receptor impedes prostate lobe-specific growth and function. Endocrinology, 148, 2264–2272.

    Article  CAS  PubMed  Google Scholar 

  28. Notini, A.J., McManus, J.F., Moore, A., Bouxsein, M., Jimenez, M., Chiu, W.S.M., Glatt, V., Kream, B.E., Handelsman, D.J., Morris, H.A., Zajac, J.D., and Davey, R.A. (2007) Osteoblast deletion of exon 3 of the androgen receptor gene results in trabecu-lar bone loss in adult male mice. Journal of Bone and Mineral Research, 22, 347–356.

    Article  CAS  PubMed  Google Scholar 

  29. Deng, C.X. and Capecchi, M.R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Molecular and Cellular Biology, 12, 3365–3371.

    CAS  PubMed  Google Scholar 

  30. Te Riele, H., Maandag, E.R., and Berns,A. (1992) Highly efficient gene targeting in embryonic stem-cells through homologous recombination with isogenic dna constructs. Proceedings of the National Academy of Sciences USA, 89, 5128–5132.

    Article  CAS  Google Scholar 

  31. Holdcraft, R.W. and Braun, R.E. (2004) Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development, 131, 459–467.

    Article  CAS  PubMed  Google Scholar 

  32. Meyers, E.N., Lewandoski, M., and Martin, G.R. (1998) An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nature Genetics, 18, 136–141.

    Article  CAS  PubMed  Google Scholar 

  33. Fedorov, L.M., HaegelKronenberger, H., and Hirchenhain, J. (1997) A comparison of the germline potential of differently aged ES cell lines and their transfected descendants. Transgenic Research, 6, 223–231.

    Article  CAS  PubMed  Google Scholar 

  34. Leneuve, P. , Colnot,S., Hamard,G., Francis, F., Niwa-Kawakita, M., Giovannini, M., and Holzenberger, M. (2003) Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles. Nucleic Acids Research, 31.

    Google Scholar 

  35. Tanaka, M., Hadjantonakis, A.K., and Nagy, A. (2001) Aggregation chimeras. Combining ES cells, diploid and tetraploid embryos. Methods in Molecular Biology, 158, 135–154.

    CAS  PubMed  Google Scholar 

  36. 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. Developmental Biology, 208, 281–292.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  38. Erdmann, G., Schutz, G., and Berger, S. (2007) Inducible gene inactivation in neurons of the adult mouse forebrain. BMC Neuroscience, 8.

    Google Scholar 

  39. Berger, S., Wolfer, D.P., Selbach, O., Alter, H., Erdmann, G., Reichardt, H.M., Chepkova, A.N., Welzl, H., Haas, H.L., Lipp,H.P., and Schutz,G. (2006) Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity. Proceedings of the National Academy of Sciences USA, 103, 195–200.

    Article  CAS  Google Scholar 

  40. Indra, A.K., Warot, X., Brocard, J., Born-ert, J.M., Xiao, J.H., Chambon, P., and Metzger, D. (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recom-binase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases. Nucleic Acids Research, 27, 4324–4327.

    Article  CAS  PubMed  Google Scholar 

  41. Hayashi, S. and McMahon, A.P. (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: A tool for temporally regulated gene activation/ inactivation in the mouse. Developmental Biology, 244, 305–318.

    Article  CAS  PubMed  Google Scholar 

  42. Cochrane, R.L., Clark, S.H., Harrs, A., and Kream, B.E. (2007) Rearrangement of a conditional allele regardless of inheritance of a cre recombinase transgene. Genesis, 45, 17–20.

    Article  CAS  PubMed  Google Scholar 

  43. Eckardt, D., Theis, M., Doring, B., Spei-del, D., Willecke, K., and Ott, T. (2004) Spontaneous ectopic recombination in cell-type-specific Cre mice removes loxP-flanked marker cassettes in vivo. Genesis, 38, 159–165.

    Article  CAS  PubMed  Google Scholar 

  44. Bula, C.M., Huhtakangas, J., Olivera, C., Bishop, J.E., Norman, A.W., and Henry, H.L. (2005) Presence of a truncated form of the vitamin D receptor (VDR) in a strain of VDR-knockout mice. Endocrinology, 146, 5581–5586.

    Article  CAS  PubMed  Google Scholar 

  45. Erben, R.G., Soegiarto, D.W., Weber, K., Zeitz, U., Lieberherr, M., Gniadecki, R., Moller, G., Adamski, J., and Balling, R. (2002) Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol.Endocrinol., 16, 1524–1537.

    Article  CAS  PubMed  Google Scholar 

  46. Notini, A.J., Davey, R.A., McManus, J.F., Bate, K.L., and Zajac, J.D. (2005) Genomic actions of the androgen receptor are required for normal male sexual differentiation in a mouse model. J.Mol.Endocri-nol., 35, 547–555.

    Article  CAS  Google Scholar 

  47. Reichardt, H.M., Kaestner, K.H., Tucker-mann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P., and Schutz, G. (1998) DNA binding of the glucocorticoid receptor is not essential for survival. Cell, 93, 531–541.

    Article  CAS  PubMed  Google Scholar 

  48. Matsumoto,T., Takeyama,K.I., Sato,T., and Kato,S. (2003) Androgen receptor functions from reverse genetic models. Journal of Steroid Biochemistry and Molecular Biology, 85, 95–99.

    Article  CAS  PubMed  Google Scholar 

  49. Sato, T., Matsumoto, T., Kawano, H., Watan-abe, T., Uematsu, Y., Sekine, K., Fukuda, T., Aihara, K.I., Krust, A., Yamada, T., Naka-michi, Y., Yamamoto, Y., Nakamura, T., Yoshimura, K., Yoshizawa, T., Metzger, D., Chambon, P., and Kato, S. (2004) Brain mas-culinization requires androgen receptor function. Proceedings of the National Academy of Sciences USA, 101, 1673–1678.

    Article  CAS  Google Scholar 

  50. Van Cromphaut, S.J., Dewerchin, M., Hoenderop, J.G.J., Stockmans, I., Van Herck, E., Kato, S., Bindels, R.J.M., Col-len, D., Carmeliet, P. , Bouillon, R., and Carmeliet, G. (2001) Duodenal calcium absorption in vitamin D receptor-knockout mice: Functional and molecular aspects. Proceedings of the National Academy of Sciences USA, 98, 13324–13329.

    Article  Google Scholar 

  51. Masuyama, R., Stockmans, I., Torrekens, S., Van Looveren, R., Maes, C., Carmeliet, P. , Bouillon, R., and Carmeliet, G. (2006) Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. Journal of Clinical Investigation, 116, 3150–3159.

    Article  CAS  PubMed  Google Scholar 

  52. Dupont, S., Krust,A ., Gansmuller, A., Dierich, A., Chambon, P., and Mark, M. (2000) Effect of single and compound knockouts of estrogen receptors alpha (ER alpha) and beta (ER beta) on mouse reproductive phenotypes. Development, 127, 4277–4291.

    CAS  PubMed  Google Scholar 

  53. Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P.C., Bock, R., Klein, R., and Schutz,G. (1999) Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nature Genetics, 23, 99–103.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory for Experimental Medicine and Endocrinology, Catholic University of Leuven, Leuven, Belgium

    Karel De Gendt & Guido Verhoeven

Authors
  1. Karel De Gendt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guido Verhoeven
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. School of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK

    Iain J. McEwan

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

De Gendt, K., Verhoeven, G. (2009). Tissue-Selective Knockouts of Steroid Receptors: A Novel Paradigm in the Study of Steroid Action. In: McEwan, I.J. (eds) The Nuclear Receptor Superfamily. Methods in Molecular Biology™, vol 505. Humana Press. https://doi.org/10.1007/978-1-60327-575-0_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-575-0_14

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-574-3

  • Online ISBN: 978-1-60327-575-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation