1991 Fiscal Year Final Research Report Summary
Analysis of the mechanism of signal transduction in T cells by using gene targetin
| Project/Area Number |
02454185
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Immunology
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| Research Institution | Chiba University |
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Principal Investigator |
SAITO Takashi Div. Mol. Genetics, Center for Neurobiol. and Immunol., Sch. Med., Chiba Univ., professor, 医学部・高次機能制御研究センター・遺伝子情報分野, 教授 (50205655)
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| Project Period (FY) |
1990 – 1991
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| Keywords | T cell receptor / CD3 chain / homologous recombination / gene targeting / signal transduction / ES cells / knockout mice |
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| Research Abstract |
This project aimed to analyze the mechanism of signal transduction in T cells by establishing variant cell lines which lack important molecules involved in signalina pathway such as T cell receptor (TCR) complex or tyrosine kinases associated with TCR complex by means of in vitro gene targeting. The target molecule we chose are CD3zeta and eta chains. We have analyzed the genomic organization of the CD3eta gene and found that CD3zeta and eta chains are produced by alternative splicin, and only last exon is different. In order to construct CD3zeta or eta-targeting vector, we introduced neor gene within the zeta or eta-specific exon, respectively, and attached HSV-tk gene at the 3' end of the each vector. Each targeting vector was transfected into a T cell hybridoma and the clones bearing one mutated allele of CD3zetaor eta were established. The zeta-transfected clone expresses a diminished level of authentic zeta mRNA and zeta protein, resulting in the decrease of the cell surface TCR/CD3 complex and no response to antien-stimulation. The results indicate that CD3zeta chain is important for the assembly and transport of TCR complex and signal transduction upon antigen stimulation not by a mutant with ransom mutations but by using a zeta-specific mutant T cell line. Moreover, the results demonstrated that there must be an "active allele" for transcription, which seems to be more suspectable for homologous recombination in this case. The inactivation of this allele diminish most of the protein encoded by the targeted gene. Therefore, these results showed that in vitro gene targeting is a powerful approach for cultured cell lines to analyze the function of molecules involved in signaling pathway. On the success of in vitro CD3zeta and eta-targeting, we also established CD3zeta or eta-targeted ES cell lines. We are now preparing, CD3zeta or eta-knockout mice in vivo to analyze the function of CD3zeta and eta in T cell differentiation.
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[Publications] Kuida, K., Furutani-Seki, M., Saito, T., Kishimoto, H., Sano, K. and Tada, T.: "Post-translational attainment of allelic exclusion of T cell receptor chain in a T cell clone" Int. Immunol. 3. 75-82 (1991)
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[Publications] Ito, T., Ishibashi, K., Imai, K., Koseki, H., Ra, C., Fernandez, E., Kantake, M., Saito, T. and Taniguchi, M.: "Monoclonal antibody against murine T cell receptor Valpha14 cross-reacts with human CD3epsilon and detects disufide-linked dimeric form." Int. Immunol. 3. 991-995 (1991)
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[Publications] Punt, J. A., Kubo, R., Saito, T., Finkel, T. H., Kathiresan, S., Blank, K. J. and Hashimoto, T.: "Surface expression of a T cell receptorbeta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins" J. Exp. Med. 174. 775-783 (1991)
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[Publications] Ohno, H., Nakamura, T., Yagita, H., Okumura, K., Taniguchi, M. and Saito, T.: "Induction of negative signal through CD2 during antigen-specific T cell activation" J. Immunol. 147. 2100-2106 (1991)
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[Publications] Saito, T., Kouyama, K., Dunne, J. F., Ransom, J., Webb, D. and Ohno, H: "No correlation between expression of CD3zeta-etadimer and activation of phosphoinositied pathway" Int. Immunol.
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