| Research Abstract |
Recently, molecular neurobiological approaches for introducing foreign genes directly into mammalian brains as well as into cultured neurons have been being developed for neuroscience researches and for gene therapy of neurological disorders. Herpes simplex virus 1 (HSV-1 ; termed "HSV" here), which is a neurotropic virus, has been expected to be one of the viral vector candidates for the purposes. We first improved and refined the methodology of gene delivery using defective HSV vectors, and summarized the experimental protocol for the HSV expression system. Next we analyzed glutamate receptor (GluR) channels using the 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. In this study, we constructed the defective HSV vectors, which contain zeta1, epsilon1 or epsilon2 cDNA under the CMV promoter. High-level expression was confirmed in the rabbit skin cells infected with each HSV clone by using RT-PCR,dot blot and Western blot analyzes. We have analyzed the molecular and functional properties of recombinant receptors expressed in Vero and CHO cells, and processed in in vivo experments. Further, we constructed baculovirus clones of deleted mutants of zeta1 subunit or site-directed mutants of AMPA-selective GluRalpha1 subunit proteins, and proceeded the detailed analysis of ligand binding regions of the proteins. We also constructed HSV clones of interferon-gamma, interleukine 2, or unknown factors involved in inhibition of neuronal cell death, and in vitro and in vivo experiments using these HSV vectors have been in progress.
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