Studies on neural plasticity using transgenic mice expressing GFP-tagged PKC

1. PKC-GFP脑区特异性表达转基因小鼠的建立与分析1)在多种启动子驱动靶蛋白脑区特异性表达的控制下表达四环素反激活子（四环素transactivator， tTA）转基因小鼠的建立与分析。在这项研究中，我们分析了在神经元特异性烯醇化酶（NSE）和钙钙调蛋白依赖性激酶n （CamKII）启动子的控制下表达tTA的转基因小鼠。前者在纹状体神经元和小脑浦肯野细胞中表达tTA，后者在包括海马在内的前脑表达tTA。此外，我们培育了在fl7和PKCγ启动子控制下表达tTA的新型转基因小鼠。2) TetOp启动子控制下表达PKC- gfp的转基因小鼠我们已经培育出了在TetOp启动子控制下表达多种gfp融合PKC亚型（PKCγ、PKCε、PKCδ和PKCζ）的转基因小鼠。3) tet调控双转基因小鼠的建立。将TetOp-PKCγ-GFP小鼠与NSE-tTA或CaMKII-tTA杂交，获得PKCγ-GFP脑区特异性表达。在NSE启动子控制下，PKCγ-GFP主要在纹状体含gaba神经元和小脑浦肯野细胞中表达。与CamKn-tTA杂交后，PKCγ-GFP在嗅球和前全脑均有表达。pkc - gfp在强力霉素治疗4周后表达完全消失，停止强力霉素治疗后表达恢复。利用PKC- gfp转基因小鼠在体内状态下的PKC易位成像，制备了这些转基因小鼠的小脑切片。用双光子激光扫描显微镜观察PKC-GFP的易位。在浦肯野细胞中，PKCγ-GFP通过代谢性谷氨酸受体的激活而瞬间转移到质膜上。

1. Development an danalysis of tet-regulated transgenic mice expressing PKC-GFP in a brain region specific manner1) Development and analysis of the transgenic mice expressing tetracycline transactivator (tTA) under the control of various promoters which drive to express target proteins in a brain region specific manner.In this study, we analyze transgenic mice which express tTA under the control of neuron specific enolase (NSE) and calcium-calmodulin dependent kinase n (CamKII) promoter. The former mice expressed tTA in striatum neurons and cerebellar Purkinje cells, while the latter expressed it in anterior brain including hippocampus. In addition, we developed novel transgenic mice which express tTA under the control ofL7 and PKCγ promoter.2) Development of transgenic mice expressing PKC-GFP under the control of tetracycline-operated (TetOp) promoterWe have developed transgenic mice which can express various subtypes of GFP-fused PKC (PKCγ, PKCε, PKCδ and PKCζ) under the control ofTetOp promoter.3) Development of tet^egulated bi-transgenic mice.The TetOp-PKCγ-GFP mice was crossed with the NSE-tTA or CaMKII-tTA to obtain brain region specific expression of PKCγ-GFP. PKCγ-GFP was mainly expressed in striatum GABA-containing neuron and cerebellar Purkinje cells under the control of NSE promoter. In contrast, PKCγ-GFP was expressed in olfactory bulb and anterior whole brain when it was crossed with CamKn-tTA mice. Expression ofPKC-GFP was completely abolished by the treatment with doxycycline for 4 weeks and the expression was recovered by the cessation ofdoxycycline treatment.2. Imaging of PKC translocation in vivo state using PKC-GFP transgenic miceCerebellar slices were prepared from these transgenic mice. Translocation of PKC-GFP was visualized by 2-photon laser scanning microscope. In Purkinje cells, PKCγ-GFP was transiently translocated to the plasma membrane by the activation of metabotropic glutamate receptors.