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:

In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors

Abstract

The myelin sheath is essential for the rapid and efficient propagation of action potentials. However, our understanding of the basic molecular mechanisms that regulate myelination, demyelination and remyelination is limited. Schwann cells produce myelin in the peripheral nervous system and remain associated with the axons of peripheral neurons throughout axonal migration to the target. Owing to the intimate relationship between these cell types it is difficult to fully reproduce their function in vitro. For this reason, we developed an approach based on the injection of an engineered virus into the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach can be used as an alternative to germline transgenesis to facilitate the investigation of peripheral nerve biology in vivo. The detailed protocol, described here, requires 3 weeks to complete. In comparison with genetic modification strategies, this protocol is a fast, reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo to investigate specific molecular mechanisms.

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

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

Figure 1: Schematic overview of the viral transduction approach in the mouse peripheral nerve.
Figure 2: Key events of the sciatic nerve injection in mouse pups and adult mice.
Figure 3: Examples of different types of infected cells after adenoviral or lentiviral injection into the mouse sciatic nerve.

Similar content being viewed by others

References

  1. Sherman, D.L. & Brophy, P.J. Mechanisms of axon ensheathment and myelin growth. Nat. Rev. Neurosci. 6, 683–690 (2005).

    Article  CAS  Google Scholar 

  2. Viader, A. et al. Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function. J. Neurosci. 31, 10128–10140 (2011).

    Article  CAS  Google Scholar 

  3. Nave, K.A. Myelination and support of axonal integrity by glia. Nature 468, 244–252 (2010).

    Article  CAS  Google Scholar 

  4. Hughes, R.A. Peripheral neuropathy. BMJ 324, 466–469 (2002).

    Article  Google Scholar 

  5. Kaewkhaw, R. et al. Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue. Nat. Protoc. 7, 1996–2004 (2012).

    Article  CAS  Google Scholar 

  6. Walsh, S. & Midha, R. Practical considerations concerning the use of stem cells for peripheral nerve repair. Neurosurg. Focus 26, E2 (2009).

    Article  Google Scholar 

  7. Zacchigna, S. & Giacca, M. Chapter 20: gene therapy perspectives for nerve repair. Int. Rev. Neurobiol. 87, 381–392 (2009).

    Article  CAS  Google Scholar 

  8. Lehmann, H.C. & Höke, A. Schwann cells as a therapeutic target for peripheral neuropathies. CNS Neurol. Disord. Drug Targets 9, 801–806 (2010).

    Article  CAS  Google Scholar 

  9. Cotter, L. et al. Dlg1-PTEN interaction regulates myelin thickness to prevent damaging peripheral nerve overmyelination. Science 328, 1415–1418 (2010).

    Article  CAS  Google Scholar 

  10. Suter, U. & Scherer, S.S. Disease mechanisms in inherited neuropathies. Nat. Rev. Neurosci. 4, 714–726 (2003).

    Article  CAS  Google Scholar 

  11. Höke, A. Animal models of peripheral neuropathies. Neurotherapeutics 9, 262–269 (2012).

    Article  Google Scholar 

  12. McGoldrick, P. et al. Rodent models of amyotrophic lateral sclerosis. Biochim. Biophys. Acta 1832, 1421–1436 (2013).

    Article  CAS  Google Scholar 

  13. Davey, R.A. & MacLean, H.E. Current and future approaches using genetically modified mice in endocrine research. Am. J. Physiol. 291, E429–E438 (2006).

    CAS  Google Scholar 

  14. Williams, R.W. et al. The math of making mutant mice. Genes Brain Behav. 2, 191–200 (2003).

    Article  CAS  Google Scholar 

  15. Joung, J.K. & Sander, D.J. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).

    Article  CAS  Google Scholar 

  16. Sung, Y.H. et al. Mouse genetics: catalogue and scissors. BMB Rep. 45, 686–692 (2012).

    Article  CAS  Google Scholar 

  17. Ozcelik, M. et al. Pals1 is a major regulator of the epithelial-like polarization and the extension of the myelin sheath in peripheral nerves. J. Neurosci. 30, 4120–4131 (2010).

    Article  CAS  Google Scholar 

  18. Perrin-Tricaud, C., Rutishauser, U. & Tricaud, N. P120 catenin is required for thickening of Schwann cell myelin. Mol. Cell Neurosci. 35, 120–129 (2007).

    Article  CAS  Google Scholar 

  19. He, T.C. et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509–2514 (1998).

    Article  CAS  Google Scholar 

  20. Glatzel, M. et al. Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system. Proc. Natl. Acad. Sci. USA 97, 442–447 (2000).

    Article  CAS  Google Scholar 

  21. Guenard, V. et al. Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice. Glia 25, 165–178 (1999).

    Article  CAS  Google Scholar 

  22. Tricaud, N. et al. Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to E-cadherin. J. Neurosci. 25, 3259–3269 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge Montpellier RIO Imaging Platform and the Animal Facility of the INM for help and technical assistance. This work was supported by the European Research Council (grant no. FP7-IDEAS-ERC 311610) and an INSERM-AVENIR grant. All animal experiments were conducted in accordance with institutional (approval no. CEEA-LR-11032) and French governmental regulations.

Author information

Authors and Affiliations

Authors

Contributions

S.G., R.F. and N.T. wrote the paper. C.P.-T., S.G., R.F. and N.T. performed experiments and analyzed data. N.T. developed the protocol and supervised the project.

Corresponding author

Correspondence to Nicolas Tricaud.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gonzalez, S., Fernando, R., Perrin-Tricaud, C. et al. In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors. Nat Protoc 9, 1160–1169 (2014). https://doi.org/10.1038/nprot.2014.073

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.073

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

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