1998 Fiscal Year Final Research Report Summary
Gene expression analyses of glutamate receptor channels by using viral vectors.
| Project/Area Number |
09670160
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Yokohama City University |
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Principal Investigator |
KAWAMOTO Susumu Yokohama City University, Department of Bacteriology, Associate Professor, 医学部, 講師 (80125921)
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| Co-Investigator(Kenkyū-buntansha) |
HATTORI Satoshi Yokohama City University, Department of Anesthesiology, Instructor, 医学部, 助手 (40275037)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Glutamate receptor / NMDA receptor / Ion channel / Herpesvirus vector / Viral vector / HSV vector / Interferon / Gene therapy |
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| Research Abstract |
The herpes simplex viral (HSV-l, termed "HSV" here) vectors have the propensity to establish long-term non-toxic latency in neurons, and the virus is large, allowing several genes to be inserted and delivered to cells. We first improved and refined the methodology of gene delivery using defective HSV vectors, and summarized the experimental protocols for the HSV expression system. NMDA glutamate receptor channels consist of the two (epsilon, zeta) subfamilies and functional variations are based on the molecular multiplicity of epsilon family subunits. Next, we constructed the defective HSV vectors which contain zeta1 cDNA.High-level expression was confirmed in the rabbit skin cells infected with the HSV clone by using RT- PCR, dot blot and Western blot analyses. Time-course study indicated that the recombinant zeta1 protein (125 kDa) was detected for the first time at 3 h postinfection, and reached maximum at 24-48 h postinfection. Molecular analyses of ligand-binding domains of glutamate receptor channels were also proceeded by using baculovirus vectors. Further, defective HSV vector was engineered to express mouse interferon (IFN)-gamma for experimental gene therapy for RSV mouse glioma. Expression of mouse IFN-gamma was shown in vitro and in vivo with immunohistochemistry. This engineered vector inhibited proliferation of RSV glioma in vitro, and its intratumoral local injection caused intratumoral necrosis in vivo. The immunological effect of this vector was examined in RSV mouse glioma implantation model. The IFN-gamma gene transferred to the cells had the effect of tumor vaccination, which was suggested to be related to natural killer cellsr These results revealed that the engineered HSV vector to express IFN-gamma may be useful for gene therapy of malignant glioma through either intratumoral local injection or exogenous tumor vaccination.
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