2001 Fiscal Year Final Research Report Summary
Studies on neural plasticity using transgenic mice expressing GFP-tagged PKC
| Project/Area Number |
12680754
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Kobe University |
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Principal Investigator |
SAKAI Norio Biosignal Research Center, Kobe University, Associate Professor, バイオシグナル研究センター, 助教授 (70263407)
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| Co-Investigator(Kenkyū-buntansha) |
SAITO Naoaki Biosignal Research Center, Kobe University, Professor, バイオシグナル研究センター, 教授 (60178499)
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| Project Period (FY) |
2000 – 2001
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| Keywords | PKC / transgenio mice / phosphorylation / neural plasticity / tet-regulated / Purkinje cells |
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| Research Abstract |
1. Development an danalysis of tet-regulated transgenic mice expressing PKC-GFP in a brain region specific manner
1) 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) promoter
We 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 mice
Cerebellar 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.
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[Publications] Sumioka, K., Shirai, Y., Sakai, N., Hashimoto, T., Tanaka, C., Yamamoto, M., Takahashi, M., Ono, Y., and Saito, N.: "Induction of 55 kDA protein of PKN by ischemia/reperfusion model of rat retina"Ophthal. Vis. Sci.. 41. 29-35 (2000)
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[Publications] Shirai, Y., Sagawa, S., Kuriyama, M., Goto, K., Sakai, N. and Saito, N.: "Subtype-specific translocation of diacylglycerol kinase a and g and its correlation with protein kinase C Shirai, Y."J. Biol. Chem.. 275. 24760-24766 (2000)
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[Publications] Ohmori, S., Sakai, N., Shirai, Y., Yamamoto, H., Miyamoto, E., Shimizu, N. and Saito, N.: "Importance of PKC targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS)"J. Biol. Chem.. 275. 26449-26457 (2000)
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