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

为了理解通过N-甲基-D-天冬氨酸受体（NMDAR）的激活引起神经表型的分子机制，我们生成并分析了具有活化NMDAR的突变小鼠。我们在小鼠中开发了有条件的诱变，以便以神经特异性的方式在NMDAR激活中取代临界序列，以进行功能分析。我们创建了携带正常外显子和突变外显子的串联阵列的小鼠，该阵列被人工内含子隔开，其中正常外显子可以专门表达。正常外显子通过Cre-loxP重组删除，从而允许突变外显子的替代表达。 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 exposed 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非竞争力的NMDAR拮抗剂完全阻止了锁骨表型。我们试图开发一种识别NMDAR的取代氨基酸的特定抗体，并获得了识别NMDAR的第二个跨膜结构域的兔多克隆抗体。识别取代氨基酸的多克隆抗体的特异性仍在检查中。我们应用了一种重组抗体技术来开发针对NMDAR取代氨基酸的特异性抗体。我们为单向亚克隆建立了一种简单，快速，高效的结扎转化方法，以产生比以前的程序大量的变换物，并构建了具有大型和高复杂性的单链FV噬菌体播放库，由大约1 x 109个独立的克隆组成。到目前为止，从图书馆获得了具有高亲和力的重组抗体。正在筛选针对NMDAR的取代氨基酸的抗体。较少的