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

1. tet调控的脑区特异性表达PKC-GFP转基因小鼠的建立与分析1）不同启动子调控的脑区特异性表达目的蛋白的四环素反式激活因子（tTA）转基因小鼠的建立与分析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）启动子控制下表达tTA的转基因小鼠。前者小鼠在纹状体神经元和小脑浦肯野细胞中表达tTA，而后者在包括海马在内的前脑中表达。此外，我们还建立了一种新的转基因小鼠，该转基因小鼠在L7和PKCγ启动子的调控下表达tTA。2）四环素操纵（TetOp）启动子调控下表达PKC-GFP的转基因小鼠的建立（PKCγ，PKCε，将TetOp-PKCγ-GFP小鼠与NSE-tTA或CaMKII-GFP小鼠杂交，获得双转基因小鼠。tTA获得PKCγ-GFP的脑区特异性表达。PKCγ-GFP在NSE启动子调控下主要表达于纹状体GABA神经元和小脑浦肯野细胞。而与CamKn-tTA小鼠杂交后，PKCγ-GFP在嗅球和全脑前部表达。多西环素处理4周后，PKC-GFP的表达完全消失，停止多西环素处理后，PKC-GFP的表达恢复.用PKC-GFP转基因小鼠进行体内PKC转位的显像制备转基因小鼠小脑切片。通过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通过代谢型谷氨酸受体的激活而瞬时易位到质膜。

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.