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

使用表达 GFP 标记 PKC 的转基因小鼠进行神经可塑性研究

• 批准号: 12680754
• 负责人: SAKAI Norio
• 金额: $ 2.43万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680754/
• 关键词: PKC; transgenio mice; phosphorylation; neural plasticity; tet-regulated; Purkinje cells; プイロテインキナーゼC; 小脳プリキンエ細胞

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.

1. 以脑区特异性方式表达 PKC-GFP 的 tet 调节转基因小鼠的开发和分析 1) 在各种启动子控制下表达四环素反式激活因子 (tTA) 的转基因小鼠的开发和分析，这些启动子驱动在大脑中表达靶蛋白在本研究中，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) 启动子控制下表达 tTA 的转基因小鼠。小鼠在纹状体神经元和小脑浦肯野细胞中表达tTA，而后者在前脑包括海马中表达tTA。此外，我们开发了在L7和PKCγ启动子控制下表达tTA的新型转基因小鼠。2）表达PKC-的转基因小鼠的开发。四环素操作（TetOp）启动子控制下的 GFP 我们开发了可以表达 GFP 融合 PKC（PKCγ、 PKCε、PKCδ和PKCδ)在TetOp启动子的控制下。3)tet^调控双转基因小鼠的开发。TetOp-PKCγ-GFP小鼠与NSE-tTA或CaMKII-tTA杂交以获得PKCγ的脑区特异性表达-GFP。PKCγ-GFP主要在NSE启动子的控制下在含有GABA的神经元和小脑浦肯野细胞中表达。相反，当与CamKn-tTA小鼠杂交时，PKCγ-GFP在嗅球和前部全脑中表达。用强力霉素治疗4周后，PKC-GFP的表达完全消失，并且在停止强力霉素治疗后恢复表达。 2.使用PKC-GFP转基因小鼠对PKC易位体内状态进行成像从这些转基因小鼠制备小脑切片，进行PKC-GFP易位。通过 2 光子激光扫描显微镜观察，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至质膜。

项目成果

Takehiko Ueyama: "Constitutively active fragment of PKN in microglia/macrophage after middle cerebral artery occlusion in rats"J.Neurochem.. 79. 903-913 (2001)

Takehiko Ueyama：“大鼠大脑中动脉闭塞后小胶质细胞/巨噬细胞中 PKN 的组成性活性片段”J.Neurochem.. 79. 903-913 (2001)

Sumioka,K.: "Induction of 55 kDa PKN cleavage product by ischemia/reperfusion model in the rat retina."Invest.Opthal.Vis.Sci.,. 41. 29-35 (2000)

Sumioka,K.：“通过大鼠视网膜缺血/再灌注模型诱导 55 kDa PKN 裂解产物。”Invest.Opthal.Vis.Sci.,。

Sakai,N.: "Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity : possible mechanism underlying SET regulation by protein kinase C."Neurochem.Int.. 36. 567-579 (2000)

Sakai,N.：“肌动蛋白细胞骨架参与血清素转运蛋白 (SET) 活性的调节：蛋白激酶 C SET 调节的可能机制。”Neurochem.Int.. 36. 567-579 (2000)

Sumioka, K 他: "Induction of 55 kDa protein of PKN by ischemia/reperfusion model of rat retina"Invest.Ophthal.Vis.Sci.. 41. 29-35 (2000)

Sumioka，K等人：“通过大鼠视网膜缺血/再灌注模型诱导PKN的55kDa蛋白”Invest.Oducing.Vis.Sci..41.29-35(2000)

Kajimoto,T.: "Subtype-specific translocation of δ-PKC and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells."Mol.Cell.Biol.. (印刷中). (2001)

Kajimoto, T.：“δ-PKC 的亚型特异性易位及其在 HeLa 细胞中神经酰胺诱导的酪氨酸磷酸化的激活。”Mol.Cell.Biol..（出版中）。