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. Development an analysis 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钙 - 钙调蛋白依赖性激酶N（CAMKII）启动子。前小鼠在纹状体神经元和小脑浦肯野细胞中表达TTA，而后者则在包括海马在内的前脑中表达了TTA。此外，我们开发了新型的转基因小鼠，这些小鼠在L7和PKCγ启动子的控制下表达TTA。2）在四环素 - 稳态（TETOP）启动子控制下表达PKC-GFP的转基因小鼠开发的转基因小鼠可以开发出可以表达GFP型PKC及PKC的各种子类型的转基因小鼠， 3）TET^egulated双转基因小鼠的开发。TETOP-PKCγ-GFP小鼠与NSE-TTA或CAMKII-TTA交叉，以获得PKCγ-GFP的脑区域特异性表达。 PKCγ-GFP主要在NSE启动子控制下在含纹状体GABA的神经元和小脑Purkinje细胞中表达。相比之下，当与CAMKN-TTA小鼠交叉时，PKCγ-GFP在嗅球和整个大脑中表达。用强力霉素治疗完全消除了PKC-GFP的表达4周，并通过doxycyclycly治疗的停止回收了表达。2。使用PKC-GFP转基因MICECEREBERAR切片对PKC易位在体内态进行成像，从这些转基因小鼠中制备。 PKC-GFP的易位通过2光子激光扫描显微镜可视化。在Purkinje细胞中，通过代谢性谷氨酸受体的激活将PKCγ-GFP瞬时易位到质膜。

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）。