The elucidation of GABAergic functions using glutamate decarboxylase conditional knockout mice

GABA是一种主要的抑制性神经递质，介导中枢神经系统（CNS）突触的抑制作用。GABA在睡眠、情绪、感知和运动等大脑功能中起着重要作用。GABA是由谷氨酸脱羧酶（GAD）合成的，GAD有GAD65和GAD67两个亚型。我们生成GAD65基因敲除小鼠和GAD67基因敲除小鼠并进行分析。GAD65基因敲除小鼠对癫痫发作和情绪行为的易感性增加，而GAD67基因敲除小鼠死于腭裂。由于GAD67常规敲除小鼠会导致新生儿死亡，因此我们无法分析GAD67基因在体内的功能，特别是在成年小鼠中的功能。因此，我们旨在利用四环素诱导系统培养GAD67条件敲除小鼠。四环素诱导系统由(1)四环素控制激活子（tTA）和(2)tetO启动子组成，用于控制tTA与tetO结合后下游基因的转录。我们试图产生更多的GAD67-tTA2敲入小鼠，其中tTA2在内源性GAD67启动子的控制下表达。GAD67- tta2敲入小鼠是GAD67条件敲除小鼠所必需的。我们构建了一个靶向载体，将其电穿孔到胚胎干细胞中，并通过Southern blot分析筛选胚胎干细胞克隆。我们鉴定了多个同源重组ES克隆。这些ES克隆将使我们能够获得GAD67-tTA2敲入小鼠。为了评估GABA在味觉功能中的作用，我们分析了GAD65基因敲除小鼠。GAD65基因敲除小鼠和野生型小鼠对四种基本口味的偏好/厌恶反应没有差异，而GAD65基因敲除小鼠比野生型小鼠消耗更少的蔗糖奎宁。这些结果表明，gad65产生的GABA与基本的味觉功能无关，而与更复杂的味觉信息加工有关。GABA不仅存在于中枢神经系统中，也存在于一些非神经元组织中。我们发现附睾狭窄细胞和胸腺髓质上皮细胞是gaba阳性细胞。少

GABA, a principal inhibitory neurotransmitter mediates inhibitory actions at central nervous system (CNS) synapses. GABA plays important roles in brain functions such as sleep, emotion, perception and movement. GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, GAD65 and GAD67. We generated and analyzed GAD65 knockout mice and GAD67 knockout mice. GAD65 knockout mice showed an increase in susceptibility to seizures and changes in emotional behaviors, whereas GAD67 knockout mice died of cleft palate.Because GAD67 conventional knockout mice resulted in a neonatal lethality, we cannot analyze GAD67 gene function in vivo, in particular in adult mice. Therefore, we aimed to develop GAD67 conditional knockout mice using tetracycline-inducible system. The tetracycline-inducible system is composed of (1) the tetracycline-controlled activator (tTA) and (2) tetO promoter to control the transcription of a downstream gene following tTA binding to tetO. We tried to genera … More te GAD67-tTA2 knock-in mice, in which tTA2 was expressed under the control of endogenous GAD67 promoter. The GAD67-tTA2 knock-in mice are necessary for GAD67 conditional knockout mice. We constructed a targeting vecor, electroporated it into ES cells, and screened ES clones by Southern blot analyses. We identified multiple homologous recombinant ES clones. These ES clones will enable us to obtain GAD67-tTA2 knock-in mice.To assess the roles of GABA in taste functions, we analyzed GAD65 knockout mice. Preference/aversion responses to four basic tastes were not different between GAD65 knockout mice and wild-type mice, while GAD65 knockout mice consumed less sucrose-quinine than did wild-type mice. These results suggest that GAD65-generated GABA is not implicated in basic taste functions, but in more complex processing of taste information.GABA is present not only in CNS but also in several nonneuronal tissues. We demonstrated that narrow cells in the epididymis and epithelial cells in the thymic medulla were GABA-positive cells. Less