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Molecular analysis of gene expression and its regulation of glutamate receptor.

基因表达及其谷氨酸受体调控的分子分析。

基本信息

批准号：
06680621
负责人：
KAWAMOTO Susumu
金额：
$ 1.41万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06680621/
关键词：
Glutamate receptor NMDA receptor Ion channel Herpesvirus vector Viral vector HSV vector Gene expression Gene therapy

项目摘要

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.

最近，将外源基因直接导入哺乳动物大脑和培养的神经元的分子神经生物学方法已经被开发出来用于神经科学研究和神经疾病的基因治疗。单纯疱疹病毒1型(HSV-1；这里称为HSV)是一种嗜神经性病毒，被认为是实现这一目的的候选病毒载体之一。我们首先改进和完善了利用缺陷HSV载体进行基因传递的方法学，并总结了HSV表达系统的实验方案。接下来，我们利用表达系统对谷氨酸受体(GluR)通道进行了分析。NMDA谷氨酸受体通道由两个(epsilon，Zeta)亚家族组成，功能变异基于epsilon家族亚单位的分子多样性。在本研究中，我们构建了CMV启动子下含有zeta1、epsilon1或epsilon2基因的缺陷型HSV载体。RT-PCR、斑点印迹和Western印迹分析证实，各HSV克隆感染的兔皮肤细胞中均有高水平表达。我们分析了在Vero和CHO细胞中表达的重组受体的分子和功能特性，并进行了体内实验。进一步，我们构建了Zeta1亚基缺失突变体和AMPA选择性GluRalpha1亚基蛋白定点突变体的杆状病毒克隆，并对蛋白质的配体结合区进行了详细的分析。我们还构建了干扰素-γ、白细胞介素2或参与抑制神经细胞死亡的未知因子的HSV克隆，并使用这些HSV载体进行了体外和体内实验。