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

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，我们生成并分析了具有活化NMDAR的突变小鼠。我们在小鼠中开发了一种条件诱变，以神经元特异性的方式替换NMDAR激活的关键序列，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串联阵列的小鼠，由人工内含子分隔，其中正常外显子可以完全表达。通过Cre-loxP重组删除正常外显子，允许突变外显子的替代表达。通过替换NMDAR第二跨膜结构域的一个关键氨基酸，我们成功地产生了具有异常NMDAR激活的突变小鼠，并且突变小鼠表现出四肢的扣合。为了阻断突变小鼠的锁扣表型，我们筛选了NMDAR激动剂、NMDAR拮抗剂和几种帕金森病药物，发现一种非竞争性NMDAR拮抗剂完全阻断了锁扣表型。我们试图建立识别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研究中。我们利用重组抗体技术，开发了一种针对NMDAR取代氨基酸的特异性抗体。我们建立了一种简单、快速、高效的单向亚克隆连接-转化方法，产生的转化体数量远远超过以往的方法，并构建了由约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。目前已从该文库中获得了一个高亲和力的重组抗体。目前正在筛选一种针对NMDAR取代氨基酸的抗体。少

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