Abstract
RNA interference (RNAi) is an intracellular mechanism for silencing gene expression utilizing short fragments of double-strand RNA that are complementary to the target messenger RNA. This gene silencing technique has now become an invaluable research tool due to its specific and strong repressive effect on a target transcript. We have recently applied a retrovirus-based RNAi system to investigate the in vivo role of the mammalian target of rapamycin (mTOR) in antigen-specific CD8 T cells, and have found that mTOR regulates memory CD8 T-cell differentiation. Here, we provide a detailed protocol for knocking down mTOR and its related molecules (raptor and FKBP12) in antigen-specific CD8 T cells. In our protocol, a mouse model of lymphocytic choriomeningitis virus infection is used, but the methods can be extended to other viral and bacterial infections as well as vaccinations. Also, the similar approach can be applied to analysis of CD4 T-cell responses.
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Acknowledgments
The authors would like to thank Professor Rafi Ahmed for providing financial support to establish this retrovirus-based RNAi system; Dr. Ben Youngblood for critical reading of our manuscript; and Dr. William Hahn for providing the pMKO.1 GFP vector.
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Araki, K., Konieczny, B.T. (2012). Utilizing a Retroviral RNAi System to Investigate In Vivo mTOR Functions in T Cells. In: Weichhart, T. (eds) mTOR. Methods in Molecular Biology, vol 821. Humana Press. https://doi.org/10.1007/978-1-61779-430-8_19
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DOI: https://doi.org/10.1007/978-1-61779-430-8_19
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