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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adaptor Proteins, Signal Transducing
-
Animals
-
CD8-Positive T-Lymphocytes / cytology
-
CD8-Positive T-Lymphocytes / immunology*
-
Carrier Proteins / genetics
-
Carrier Proteins / metabolism
-
Cell Differentiation
-
Cell Line
-
Disease Models, Animal
-
Flow Cytometry
-
Gene Expression Regulation
-
Gene Knockdown Techniques*
-
Gene Silencing / immunology
-
Genetic Vectors
-
Humans
-
Immunity, Innate / genetics*
-
Lymphocytic Choriomeningitis / genetics
-
Lymphocytic Choriomeningitis / metabolism
-
Lymphocytic Choriomeningitis / virology
-
Mice
-
Mice, Inbred C57BL
-
RNA Interference*
-
Regulatory-Associated Protein of mTOR
-
Retroviridae
-
TOR Serine-Threonine Kinases / genetics*
-
TOR Serine-Threonine Kinases / immunology*
-
Tacrolimus Binding Protein 1A / genetics
-
Tacrolimus Binding Protein 1A / metabolism
-
Transduction, Genetic
Substances
-
Adaptor Proteins, Signal Transducing
-
Carrier Proteins
-
Regulatory-Associated Protein of mTOR
-
Rptor protein, mouse
-
TOR Serine-Threonine Kinases
-
Tacrolimus Binding Protein 1A