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:

Generation of T-cell receptor retrogenic mice

Abstract

T-cell receptor (TCR) transgenic (Tg) mice have revolutionized our understanding of many aspects of T-cell biology. Whereas they provide an almost unlimited source of T cells with a single specificity, breeding them onto different backgrounds and/or new knockout/knock-in mouse models is often time-consuming (6 months to several years), which can make the process costly and can significantly delay research. This protocol describes a new method for expressing defined TCR-α and TCR-β proteins from a single 2A peptide–linked multicistronic retroviral vector in mice, using retrovirus-mediated stem cell gene transfer. We refer to these as 'retrogenic' (Rg) mice ('retro' from retrovirus and 'genic' from Tg) to avoid confusion with traditional transgenic mice. We have successfully used this approach to express over 50 different TCRs on several different mouse backgrounds in as little as 6 weeks.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Flow chart of steps required to make TCR-2A vectors, generate retroviral producers and create TCR retrogenic mice.
Figure 2: Vector maps for (a) pMIGII and (b) pEQ-Pam3(-E).
Figure 3: Details of oligonucleotide primers used to generate 2A-linked TCR vectors.
Figure 4: Flow cytometric analysis of TCR Rg mice.

Similar content being viewed by others

References

  1. de Felipe, P. Polycistronic viral vectors. Curr. Gene Ther. 2, 355–378 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. de Felipe, P. Skipping the co-expression problem: the new 2A “CHYSEL” technology. Genet. Vaccines Ther. 2, 13 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  3. de Felipe, P. et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol. 24, 68–75 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Szymczak, A.L. & Vignali, D.A. Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin. Biol. Ther. 5, 627–638 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Ryan, M.D. & Drew, J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928–933 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Donnelly, M.L. et al. Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J. Gen. Virol. 82, 1013–1025 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Miller, J.F. & Flavell, R.A. T-cell tolerance and autoimmunity in transgenic models of central and peripheral tolerance. Curr. Opin. Immunol. 6, 892–899 (1994).

    Article  CAS  PubMed  Google Scholar 

  9. Benoist, C. & Mathis, D. Positive and negative selection of the T cell repertoire in MHC class II transgenic mice. Semin. Immunol. 1, 117–124 (1989).

    CAS  PubMed  Google Scholar 

  10. Arnold, P.Y., Burton, A.R. & Vignali, D.A. Diabetes incidence is unaltered in glutamate decarboxylase 65-specific TCR retrogenic nonobese diabetic mice: generation by retroviral-mediated stem cell gene transfer. J. Immunol. 173, 3103–3111 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Holst, J., Vignali, K.M., Burton, A.R. & Vignali, D.A.A. Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat. Methods 3, 191–197 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Baldwin, T.A., Sandau, M.M., Jameson, S.C. & Hogquist, K.A. The timing of TCR alpha expression critically influences T cell development and selection. J. Exp. Med. 202, 111–121 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Szymczak-Workman, A.L., Vignali, K.M. & Vignali, D.A.A. Generation of 2A peptide-linked multicistronic vectors. in DNA Delivery/Gene Transfer Lab Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, in the press).

  14. Szymczak, A.L. et al. The CD3-ε proline-rich sequence, and its interaction with Nck, is not required for T cell development and function. J. Immunol. 175, 270–275 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Persons, D.A., Mehaffey, M.G., Kaleko, M., Nienhuis, A.W. & Vanin, E.F. An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol. Dis. 24, 167–182 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Persons, D.A. et al. Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. Blood 90, 1777–1786 (1997).

    CAS  PubMed  Google Scholar 

  17. Hawley, R.G., Lieu, F.H., Fong, A.Z. & Hawley, T.S. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1, 136–138 (1994).

    CAS  PubMed  Google Scholar 

  18. Persons, D.A. et al. Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells. Nat. Med. 4, 1201–1205 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Rees, W.A., Yager, T.D., Korte, J. & von Hippel, P.H. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32, 137–144 (1993).

    Article  CAS  PubMed  Google Scholar 

  20. Varadaraj, K. & Skinner, D.M. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene 140, 1–5 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Baskaran, N. et al. Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6, 633–638 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Henke, W., Herdel, K., Jung, K., Schnorr, D. & Loening, S.A. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957–3958 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to everyone in the Vignali lab for helping this protocol evolve into its current form. This work was supported by the NIH (AI52199, AI39480), the Juvenile Diabetes Research Foundation International (1-2004-141), a Cancer Center Support CORE grant (CA-21765) and the American Lebanese Syrian Associated Charities (ALSAC).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dario A A Vignali.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Holst, J., Szymczak-Workman, A., Vignali, K. et al. Generation of T-cell receptor retrogenic mice. Nat Protoc 1, 406–417 (2006). https://doi.org/10.1038/nprot.2006.61

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.61

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