Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Xenopus laevis transgenesis by sperm nuclear injection

Nature Protocols volume 1, pages 2195–2203 (2006)Cite this article

Abstract

The stable integration of transgenes into embryos of the frog Xenopus laevis is achieved using the procedure described here. Linear DNA containing the transgene is incorporated randomly into sperm nuclei that have had their membranes disrupted with detergent treatment. Microinjection of these nuclei into unfertilized eggs produces viable embryos that can be screened for activity of the transgene. The proportion of embryos that harbor the transgene varies from 10 to 40% of the total number of surviving embryos. Multiple copies of the transgene can integrate as a concatemer into the sperm genome, and more than one site of DNA integration might occur within resulting animals. Germ cell transmission of the transgene is routine and the procedure is well suited to the production of transgenic reporter frog lines. One day should be allocated for the preparation of the sperm nuclei, which are stored as aliquots for future use. The transgenesis reaction and egg injection take one morning.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of GFP in two distinct myeloid cell transgenic Xenopus lines.
Figure 2: Injection apparatus.
Figure 3: Counting sperm nuclei in the hemocytometer.
Figure 4: Injection dish and needle-holder.
Figure 5: Selection of fertilized Xenopus embryos.

Similar content being viewed by others

References

  1. Kroll, K.L. & Amaya, E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173–3183 (1996).

    CAS  PubMed  Google Scholar 

  2. Sive, H.L., Grainger, R.M. & Harland, R.M. Early development of Xenopus laevis. A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

    Google Scholar 

  3. Sparrow, D.B., Latinkic, B. & Mohun, T.J. A simplified method of generating transgenic Xenopus. Nucleic Acids Res. 28, E12 (2000).

    Article  CAS  Google Scholar 

  4. Allen, B.G. & Weeks, D.L. Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. Nat. Methods 2, 975–979 (2005).

    Article  CAS  Google Scholar 

  5. Allen, B.G. & Weeks, D.L. Using phiC31 integrase to create transgenic Xenopus laevis. Nature Protocols 1 (3): 1247–1257 (2006).

    Article  Google Scholar 

  6. Pan, F.C., Chen, Y., Loeber, J., Henningfeld, K. & Pieler, T. I-SceI meganuclease-mediated transgenesis in Xenopus. Dev. Dyn. 235, 247–252 (2006).

    Article  Google Scholar 

  7. Ogino, H., McConnell, W.B. & Grainger, R.M. Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease. Mech. Dev. 123, 103–113 (2006).

    Article  CAS  Google Scholar 

  8. Hajime, O., McConnell, W.B. & Grainger, R.M. High-throughput transgenesis in xenopus using I-Sce1 meganuclease. Nat. Protoc. (doi: 10.1038/nprot.2006.208).

  9. Smith, S.J. & Mohun, T.J. Frog transgenesis made simple. Nat. Methods 2, 897–898 (2005).

    Article  CAS  Google Scholar 

  10. Latinkic, B.V. et al. Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos. Dev. Biol. 245, 57–70 (2002).

    Article  CAS  Google Scholar 

  11. Latinkic, B.V. et al. Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus embryonic development. Development 131, 669–679 (2004).

    Article  CAS  Google Scholar 

  12. Karaulanov, E., Knochel, W. & Niehrs, C. Transcriptional regulation of BMP4 synexpression in transgenic Xenopus. Embo. J. 23, 844–856 (2004).

    Article  CAS  Google Scholar 

  13. Allende, M.L., Manzanares, M., Tena, J.J., Feijoo, C.G. & Gomez-Skarmeta, J.L. Cracking the genome's second code: enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos. Methods 39, 212–219 (2006).

    Article  CAS  Google Scholar 

  14. Sparrow, D.B. et al. Regulation of the tinman homologues in Xenopus embryos. Dev. Biol. 227, 65–79 (2000).

    Article  CAS  Google Scholar 

  15. Smith, S.J., Kotecha, S., Towers, N., Latinkic, B.V. & Mohun, T.J. XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus. Mech. Dev. 117, 173–186 (2002).

    Article  CAS  Google Scholar 

  16. Smith, S.J. et al. The MLC1v gene provides a transgenic marker of myocardium formation within developing chambers of the Xenopus heart. Dev. Dyn. 232, 1003–1012 (2005).

    Article  CAS  Google Scholar 

  17. Breckenridge, R.A., Mohun, T.J. & Amaya, E. A role for BMP signalling in heart looping morphogenesis in Xenopus. Dev. Biol. 232, 191–203 (2001).

    Article  CAS  Google Scholar 

  18. Brown, D.D. et al. Thyroid hormone controls multiple independent programs required for limb development in Xenopus laevis metamorphosis. Proc. Natl. Acad. Sci. USA 102, 12455–12458 (2005).

    Article  CAS  Google Scholar 

  19. Gilchrist, M.J. et al. Defining a large set of full-length clones from a Xenopus tropicalis EST project. Dev. Biol. 271, 498–516 (2004).

    Article  Google Scholar 

  20. Du Pasquier, D. et al. Survivin increased vascular development during Xenopus ontogenesis. Differentiation 74, 244–253 (2006).

    Article  CAS  Google Scholar 

  21. Li, M. & Rohrer, B. Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis. Cell Res. 16, 99–105 (2006).

    Article  CAS  Google Scholar 

  22. Chae, J., Zimmerman, L.B. & Grainger, R.M. Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines. Mech. Dev. 117, 235–241 (2002).

    Article  CAS  Google Scholar 

  23. Ryffel, G.U. et al. Tagging muscle cell lineages in development and tail regeneration using Cre recombinase in transgenic Xenopus. Nucleic Acids Res. 31, e44 (2003).

    Article  Google Scholar 

  24. Bronchain, O.J., Hartley, K.O. & Amaya, E. A gene trap approach in Xenopus. Curr. Biol. 9, 1195–1198 (1999).

    Article  CAS  Google Scholar 

  25. Huang, H., Marsh-Armstrong, N. & Brown, D.D. Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase. Proc. Natl. Acad. Sci. USA 96, 962–967 (1999).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Stuart J Smith, Lynne Fairclough & Timothy J Mohun

  2. School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, UK

    Branko V Latinkic

  3. Developmental Biology Program, Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, 2010, NSW, Australia

    Duncan B Sparrow

Authors
  1. Stuart J Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lynne Fairclough
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Branko V Latinkic
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Duncan B Sparrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Timothy J Mohun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Timothy J Mohun.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Procedure to fill an injection needle (step 30) (MOV 9556 kb)

Supplementary Video 2

Injection technique (MOV 9609 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smith, S., Fairclough, L., Latinkic, B. et al. Xenopus laevis transgenesis by sperm nuclear injection. Nat Protoc 1, 2195–2203 (2006). https://doi.org/10.1038/nprot.2006.325

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.325

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing