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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，我们培育并分析了携带激活NMDAR的突变小鼠。我们在小鼠身上开发了一种条件突变，以神经元特异性的方式替换NMDAR激活中的关键序列，以进行功能分析。我们创造了携带正常外显子和突变外显子的串联阵列的小鼠，这些外显子由人工内含子分隔，其中正常外显子可以唯一表达。Cre-loxP重组删除了正常外显子，允许突变外显子的替代表达。我们成功地通过在NMDAR的第二跨膜结构域中替换一个关键氨基酸来产生具有异常NMDAR激活的突变小鼠，并且突变小鼠表现出肢体的紧握。为了阻止突变小鼠的紧握表型，我们筛选了NMDAR激动剂、NMDAR拮抗剂和几种药物…并发现非竞争性的NMDAR拮抗剂完全阻断了NMDAR的紧握表型。我们试图开发识别NMDAR取代氨基酸的特异性抗体，并获得识别NMDAR第二跨膜结构域的兔多克隆抗体。识别被取代氨基酸的多克隆抗体的特异性仍在检验中。我们应用重组抗体技术研制了针对NMDAR被取代氨基酸的特异性抗体。我们建立了一种简单、快速、高效的单向亚克隆连接转化方法，产生的转化子数量远远超过以往的方法，并构建了由大约1×109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。到目前为止，从文库中获得了一株高亲和力的重组抗体。针对NMDAR的取代氨基酸的抗体正在筛选中。较少