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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
de Felipe, P. Polycistronic viral vectors. Curr. Gene Ther. 2, 355–378 (2002).
de Felipe, P. Skipping the co-expression problem: the new 2A “CHYSEL” technology. Genet. Vaccines Ther. 2, 13 (2004).
de Felipe, P. et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol. 24, 68–75 (2006).
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).
Ryan, M.D. & Drew, J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928–933 (1994).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Baskaran, N. et al. Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6, 633–638 (1996).
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).
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).
The authors declare no competing financial interests.
About this article
Scientific Reports (2019)
Genetically modified hematopoietic stem/progenitor cells that produce IL-10–secreting regulatory T cells
Proceedings of the National Academy of Sciences (2019)
Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling
Science Signaling (2019)
Iranian Journal of Science and Technology, Transactions of Electrical Engineering (2019)
Nature Communications (2019)