Investigation of signal transduction in cerebellar Purkinje cell by in vivo expression of pertusis toxin.

为了研究三量GTP结合蛋白在小脑浦肯野细胞中的作用，我们制备了两种不同的转基因小鼠。首先，我们培育了在浦肯野细胞中表达百日咳毒素（PTX）的转基因小鼠，通过Gi/o家族抑制百日咳毒素信号。其次，我们产生了缺乏RGS8的突变小鼠，RGS8具有Gi/o家族的GTPase激活结构域，在浦肯野细胞中显著表达。我们通过显微注射浦肯野细胞特异性L7启动子驱动的PTX基因，获得了L7-PTX转基因小鼠。一只L7-PTX转基因小鼠在小脑中表达PTX mRNA，自发性运动减少。转基因小鼠在7周大时死亡。其他转基因小鼠在小脑中表达少量PTX mRNA，转基因未传递给后代。这些结果表明，PTX在小脑中的表达导致生存能力降低。为了克服这种情况，我们制作了一种多西环素抑制PTX表达的转基因载体。我们通过基因靶向的方法产生了RGS8敲除小鼠（KO）。我们在两个FRT位点两侧引入了loxP位点和新霉素耐药基因。我们生成了携带loxP序列外显子完整的RGS8突变体，以及两个外显子缺失的RGS8 KO小鼠。RGS8 KO运动协调、自主运动和体重正常。然而，RGS8 KO的体温低于野生型幼崽。此外，在RGS8 KO小鼠中模拟GABAB受体激动剂巴氯芬诱导的低温。这些结果表明RGS8通过控制GABAB受体信号通路调节体温。

In order to investigate the role of trimetric GTP binding proteins in cerebellar Purkinje cell, we generated two different kind of transgenic mice.First we generated transgenic mice which express pertusis toxin (PTX) in Purkinje cells to inhibit the signal via a Gi/o family. Secondly, we generated mutant mice which lack RGS8 that has a GTPase activating domein for Gi/o family and is expressed prominently in Purkinje cells. We generated L7-PTX transgenic mice by microinjection of PTX gene driven by Purkinje cell specific L7 promoter. One L7-PTX transgenic mouse expressed PTX mRNA in cerebellum and showed reduction in spontaneous movement. The transgenic mouse died at 7 weeks old. Other transgenic mice expressed little PTX mRNA in the cerebellum and transgene were not transmitted to the offspring. These results suggested that expression of PTX in the cerebellum results in reduced viability. To overcome this situation, we generated a transgenic vector in which PTX expression was suppressed by doxycycline. We generated RGS8 knockout mice (KO) by gene targeting. We introduced loxP sites and neomycin resistant gene flanked by two FRT sites. We generated RGS8 mutant carrying loxP sequence with intact exons as well as the RGS8 KO mice in which two exons were deleted. RGS8 KO showed normal motor coordination, spontaneous movement and body weight. However, body temperature of RGS8 KO was lower than that of wild-type littermate. Furthermore, hypothermia induced by GABAB receptor agonist baclofen was mimicked in RGS8 KO mice. These results suggested that RGS8 regulates body temperature by controlling GABAB receptor signaling.