Analysis of function of NMDA receptor using genetically engineered mice by conditional mutagenesis

利用基因工程小鼠条件诱变分析 NMDA 受体功能

基本信息

批准号：
15500277
负责人：
SASAOKA Toshikuni
金额：
$ 2.11万
依托单位：
NATIONAL INSTITUTE FOR BASIC BIOLOGY (2004)Okazaki National Research Institutes (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

To understand molecular mechanism causing the neurological phenotype through an activation of the N-methyl-D-aspartate receptor (NMDAR), we generated and analyzed mutant mice harboring activated NMDAR. We developed a conditional mutagenesis in mice to enable substitution of a critical sequence in NMDAR activation in a neuron-specific manner for purposes of functional analysis. We created mice carrying a tandem array of a normal exon and a mutated exon, separated by an artificial intron, in which the normal exon could be exclusively expressed. The normal exon was deleted by Cre-loxP recombination, allowing the alternative expression of the mutant exon. We successfully generated the mutant mice harboring an aberrant NMDAR activation by a substitution of a critical amino acid in the second trans-membrane domain of the NMDAR, and the mutant mice exhibited a clasping of the limbs.To block the clasping phenotype of the mutant mice we screened NMDAR agonists, NMDAR antagonists and several dru … More gs for Parkinson's disease and found a non-competitive NMDAR antagonist completely blocked the clasping phenotype.We tried to develop a specific antibody recognizing the substituted amino acid of the NMDAR and obtained a rabbit polyclonal antibody recognizing the second trans-membrane domain of the NMDAR. The specificity of the polyclonal antibody recognizing the substituted amino acid is still being examined.We applied a recombinant antibody technology to develop a specific antibody against the substituted amino acid of the NMDAR. We established a simple, rapid and highly efficient ligation-transformation method for unidirectional subcloning to generate far larger numbers of transformants than previous procedures, and constructed single chain Fv phage-display libraries with a large-scale and a high-complexity consisting of approximately 1 x 109 independent clones. So far a recombinant antibody with a high affinity was obtained from the library. An antibody against the substituted amino acid of the NMDAR is being screened. Less

为了了解通过激活 N-甲基-D-天冬氨酸受体 (NMDAR) 导致神经表型的分子机制，我们生成并分析了携带激活 NMDAR 的突变小鼠。我们通过人工内含子创建了携带正常外显子和突变外显子的串联阵列的小鼠，其中正常外显子可以被专门表达。通过 Cre-loxP 重组删除了外显子，从而允许突变外显子的替代表达，通过替换 NMDAR 的第二个跨膜结构域中的关键氨基酸，我们成功地生成了具有异常 NMDAR 激活的突变小鼠。突变小鼠表现出四肢紧握。为了阻止突变小鼠的紧握表型，我们筛选了 NMDAR 激动剂、NMDAR 拮抗剂和几种治疗帕金森病的药物我们试图开发识别NMDAR取代氨基酸的特异性抗体，并获得了识别NMDAR第二跨膜结构域的特异性兔多克隆抗体。识别取代氨基酸的多克隆抗体仍在研究中。我们应用重组抗体技术开发了针对NMDAR取代氨基酸的特异性抗体。我们建立了一种简单、快速、高效的抗体。高效的单向亚克隆连接转化方法产生的转化子数量远多于以前的方法，并构建了迄今为止由约 1 x 109 个独立克隆组成的大规模、高复杂性的单链 Fv 噬菌体展示文库。从文库中获得了具有高亲和力的重组抗体，正在筛选针对 NMDAR 取代氨基酸的抗体。