Paired cloning of the T cell receptor alpha and beta genes from a single T cell without the establishment of a T cell clone.
T cell receptors, which recognize antigen peptides on MHC molecules, are essential probes for the analysis of T cell antigen specificity. The identification of paired T cell receptor (TCR) chains, alpha/beta or gamma/delta, usually requires the establishment of T cell clones, which is not always available. In this study, we tried, as an alternative method, the paired cloning of TCR alpha/beta genes directly from a single T cell. T cells were sorted as a single cell from which RNA was extracted. Then, TCR alpha/beta CDR3 regions were amplified from the single cell-derived cDNA by reverse transcriptase-polymerase chain reaction to determine their sequences. We successfully identified pairs of TCR alpha/beta genes, and reconstructed the TCR molecule by a bacterial expression system. This strategy makes it possible to obtain recombinant TCR molecules from a single T cell without cellular cloning and promotes the investigation of T cell antigen specificity.
Kurokawa
M
M
Rheumatology, Immunology and Genetic Program, Institute of Medical Science, St Marianna University School of Medicine, Kawasaki, Japan. manae@marianna-u.ac.jp
Tong
J
J
Matsui
T
T
Masuko-Hongo
K
K
Yabe
T
T
Nishioka
K
K
Yamamoto
K
K
Kato
T
T
D002454:Cell Differentiation; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D016693:Receptors, Antigen, T-Cell, alpha-beta