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:

Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo

Nature Protocols volume 1, pages 3049–3055 (2006)Cite this article

Abstract

Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult floxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efficient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.

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: A schematic structure of the recombinant retroviral genome of CAG-GFP/cre.
Figure 2
Figure 3: Stereotaxic injection.
Figure 4: New neurons expressing GFP/cre in the adult dentate gyrus.

Similar content being viewed by others

References

  1. Gage, F.H. Mammalian neural stem cells. Science 287, 1433–1438 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Ming, G.L. & Song, H. Adult neurogenesis in the mammalian central nervous system. Annu. Rev. Neurosci. 28, 223–250 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Tashiro, A., Sandler, V.M., Toni, N., Zhao, C. & Gage, F.H. NMDA-receptor-mediated, cell-specific integration of new neurons in adult dentate gyrus. Nature 442, 929–933 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Tsien, J.Z. et al. Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87, 1317–1326 (1996).

    Article  CAS  PubMed  Google Scholar 

  5. van Praag, H. et al. Functional neurogenesis in the adult hippocampus. Nature 415, 1030–1034 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Zhao, C., Teng, E.M., Summers, R.G. Jr . Ming, G.L. & Gage, F.H. Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J. Neurosci. 26, 3–11 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ge, S. et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 439, 589–593 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Soneoka, Y. et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Burns, J.C., Friedmann, T., Driever, W., Burrascano, M. & Yee, J.K. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033–8037 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Naviaux, R.K., Costanzi, E., Haas, M. & Verma, I.M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kaspar, B.K. et al. Adeno-associated virus effectively mediates conditional gene modification in the brain. Proc. Natl. Acad. Sci. USA 99, 2320–2325 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zufferey, R., Donello, J.E., Trono, D. & Hope, T.J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kafri, T., van Praag, H., Gage, F.H. & Verma, I.M. Lentiviral vectors: regulated gene expression. Mol. Ther. 1, 516–521 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Klein, S.A. et al. Comparison of TagMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication. J. Virol. Methods 107, 168–175 (2003).

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Ayumu Tashiro

    Present address: Centre for the Biology of Memory, Norwegian University of Science and Technology, Medical-Technical Research Centre, NO-7489, Trondheim, Norway

Authors and Affiliations

  1. Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037, California, USA

    Ayumu Tashiro, Chunmei Zhao & Fred H Gage

Authors
  1. Ayumu Tashiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chunmei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fred H Gage
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fred H Gage.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tashiro, A., Zhao, C. & Gage, F. Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo. Nat Protoc 1, 3049–3055 (2006). https://doi.org/10.1038/nprot.2006.473

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.473

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