Studies on neural calcium signaling using knock-out mice

我们计划引入P/Q型和N型电压门控性钙通道基因的靶向阻断，但未能达到目的。为了生产钙通道基因敲除小鼠，我们不仅在建立同源重组ES细胞系方面遇到困难，而且在通过注射ES细胞制备嵌合小鼠方面也遇到困难。我们不知道为什么会遇到这么多困难。但考虑到目前还没有人成功敲除钙通道基因的情况，这些基因可能存在某种特定的调控机制。同时，我们继续研究了钙通道在离子传导和激活中的功能特性。我们还对编码一种新的钙通道的cDNA进行了分子克隆，这种钙通道不是电压依赖性的，而是通过未鉴定的细胞内信使被细胞表面受体激活的。我们获得了几个cDNA，我们现在正在使用重组表达系统来表征它们的功能特性。一个项目，以淘汰这一渠道的家庭之一，是在进行中。我们已经建立了同源重组的ES细胞系，并期望在不久的将来获得嵌合小鼠，因为P/Q型钙通道基因的突变可以引起小鼠和人的小脑共济失调和其他神经系统症状，我们研究了小脑浦肯野细胞的电生理，并与我们在重组系统中获得的功能特性进行了比较。这些结果将有助于我们深入了解钙通道缺陷的病理机制。

We planned to introduce targetted disruption of the genes of P/Q type and N type voltage-gated calcium channels, but we failed to attain the goal. For producing calcium channel-knock-out mice, we had difficulties not only in establishing homologously recombinated ES cell lines but also in making chimeric mice by injecting the ES cells. It is not known why we had so many difficulties. But if we consider the current situation that no one has succeeded in knocking out calcium channel genes, there may be some specific regulatory mechanism for those genes.In meantime, we continued to characterize functional properties in ion conduction and activation of calcium channels. Also we made molecular cloning of cDNAs encoding a new entity of calcium channels that are not voltage-dependent but activated by cell surface receptors through unidentified intracellular messengers. We obtained several cDNAs, and we are now characterizing their functional properties using a recombinant expresion system. A project to knock out one of this channel family is in progress. We have already established homologously recombinated ES cell lines, and expect to have chimeric mice in near future.Because mutations in the P/Q type calcium channel gene can cause cerebellar ataxia and other neurological symptoms in mice and human, we studied cerebellar Purkinje cells electrophysiologically, and compared the functional properties with those we obtained in recombinant system. These results will give us an insight into the pathological mechanism of calcium channel defects.