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

点击翻译按钮获取中文摘要

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.