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Generation of transgenic mice expressing alphalA-calcium channel protein with highly expanded polyglutamine tract.

产生表达具有高度扩展的聚谷氨酰胺束的α1A-钙通道蛋白的转基因小鼠。

基本信息

批准号：
15590881
负责人：
ISHIKAWA Kinya
金额：
$ 2.11万
依托单位：
Tokyo Medical and Dental University
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

The most important aim of this study is to generate transgenic mice that express human alpha1A-calcium channel protein with highly expanded polyglutamine tract to gain model mice that could be a n indispensable material for exploring pathogenesis of SCA6, an autosomal dominant cerebellar ataxia caused by a mild expansion of polyglutamine in alpha1A-calcium channel. To accomplish this, we cloned human alpha1A-calcium channel cDNA which contains a CAG repeat tract encoding 165 polyglutamines. This clone was inserted downstream of the Purkinje cell specific promoter. Then the construct was microinjected into C57B16/J mouse strain. We obtained 200 mice by two microinjections, and screened by PCR amplification of genomic DNA, whether any of these mice harbor human alpha1A-calcium channel construct. However, we did not observe such mice. Knowing that similar difficulties could be overcome by changing mice strain to Fbv, we next underwent microinjection to the Fbv mice strain. These experimen … More ts were done on September 2004. After screening mice DNA for positive injection by aforementioned procedure, we found on December 2004 that two FO mice properly harbor human alpha1A-calicum channel cDNA construct. These mice were backcrossed with wild type Fbv mice. By the end of March 2005, we have screened several mice of F1 generation. However, we did not observe evidence for germline transmission. We will continue backcrossing FO mice with wild -type mice.We also planned to analyze SCA6 patients' brains whether the expanded polyglutamine induces proteolytic cleavage of alpha1A calcium channel as suggested by our preliminary in vitro study. For this aim, we generated rabbit polyclonal antibody against the polypeptide near the polyglutamine tract. The specificity of this antibody was assessed by SDS-PAGE analysis on recombinant channel protein. By using this antibody, we found that the polyglutamine aggregates seen in SCA6 Purkinje cells indeed contain alpha1A-calcium channel. We also observed that large aggregates identified by the antibody against the Carboxyl-terminal peptide are formed by cluster of polyglutamine aggregates. These findings suggested that the human alpha1A-calcium channel protein is indeed processed in Purkinje cells. Processed fragments are likely to be prone to aggregatie formation Less

本研究最重要的目的是建立表达人α1A-钙通道蛋白的转基因小鼠，获得高度扩张的聚谷氨酰胺通道，为探讨SCA6的发病机制提供必要的材料。SCA6是一种常染色体显性遗传性小脑性共济失调，由α1A-钙通道中聚谷氨酰胺的轻度扩张引起。为此，我们克隆了含有编码165个多谷氨酸的CAG重复序列的人α1A-钙通道基因。该克隆插入浦肯野细胞特异性启动子下游。将构建的重组蛋白显微注射到C57B16/J小鼠体内。我们通过两次微量注射获得了200只小鼠，并通过基因组DNA的PCR扩增进行筛选，无论这些小鼠中是否有任何一只含有人α1A-钙通道结构。但是，我们没有观察到这样的老鼠。知道可以通过将小鼠品系改为FBV来克服类似的困难，我们接下来对FBV品系进行了显微注射。这些是对…的实验2004年9月进行了更多的测试。用上述方法筛选阳性注射的小鼠DNA后，2004年12月，我们发现两只FO小鼠正确地含有人α1A-Calicum通道的cDNA构建。将这些小鼠与野生型FBV小鼠回交。截至2005年3月底，我们已经筛选出几只F1代小鼠。然而，我们没有观察到生殖系传播的证据。我们将继续与野生型小鼠回交FO小鼠。我们还计划分析SCA6患者的大脑是否会像我们的初步体外研究所建议的那样，诱导α1A钙通道的蛋白水解性切割。为此，我们在聚谷氨酰胺束附近制备了针对该多肽的兔多克隆抗体。通过对重组通道蛋白的SDS-PAGE分析，鉴定该抗体的特异性。通过使用这种抗体，我们发现在SCA6Purkinje细胞中看到的聚谷氨酰胺聚集体确实含有α1A-钙通道。我们还观察到，由针对羧基末端肽的抗体鉴定的大聚集体是由聚谷氨酰胺聚集体形成的。这些发现表明，人类α1A-钙通道蛋白确实是在浦肯野细胞中加工的。经处理的碎片很可能较少地易于聚集形成