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

本研究的最重要目的是建立表达人α 1A-钙通道蛋白的多聚谷氨酰胺通道高度扩增的转基因小鼠，为研究SCA 6（一种由α 1A-钙通道中的多聚谷氨酰胺轻度扩增引起的常染色体显性遗传性小脑共济失调）的发病机制提供不可缺少的实验材料。为了实现这一点，我们克隆了人α 1A-钙通道cDNA，其中包含一个CAG重复序列编码165聚谷氨酰胺。将该克隆插入浦肯野细胞特异性启动子的下游。然后将构建体显微注射到C57 B16/J小鼠品系中。我们通过两次显微注射获得了200只小鼠，并通过基因组DNA的PCR扩增进行筛选，这些小鼠中是否有人α 1A-钙通道构建体。然而，我们没有观察到这样的小鼠。知道类似的困难可以通过将小鼠品系改变为Fbv来克服，我们接下来对Fbv小鼠品系进行显微注射。这些实验 关于我们 2004年9月完成。通过上述方法筛选小鼠DNA阳性注射后，我们于2004年12月发现两只FO小鼠正确携带人α 1A-钙通道cDNA构建体。将这些小鼠与野生型Fbv小鼠回交。到2005年3月底，我们已经筛选出了几只F1代小鼠。然而，我们没有观察到生殖系传播的证据。我们将继续将FO小鼠与野生型小鼠进行回交，并计划分析SCA 6患者的大脑是否如我们的初步体外研究所示，扩增的多聚谷氨酰胺诱导α 1A钙通道的蛋白水解裂解。为了这个目的，我们产生了兔多克隆抗体对多肽附近的多聚谷氨酰胺道。通过对重组通道蛋白的SDS-PAGE分析来评估该抗体的特异性。通过使用这种抗体，我们发现在SCA 6浦肯野细胞中看到的聚谷氨酰胺聚集体确实含有α 1A-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由聚谷氨酰胺聚集体的簇形成。这些发现表明，人类α 1A-钙通道蛋白确实在浦肯野细胞中加工。经过处理的碎片很可能易于形成聚集体

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