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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.

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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.

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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).

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Correspondence to Dario A A Vignali.

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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

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