| Project/Area Number |
12558091
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Neuroscience in general
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
HONMA Ken-ichi Hokkaido Univ., Grad. School of Medicine, Prof., 大学院・医学研究科, 教授 (40113625)
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| Co-Investigator(Kenkyū-buntansha) |
KUBOTA Hidehiro Atto Coorperation, Research Manager, 技術開発部・学術研究科, 課長(研究職)
OHMIYA Yoshihiro National Institute of AIST, Chief researcher, グループ長 (20223951)
HONMA Sato Hokkaido Univ., Grad. School of Medicine, Asso. Prof., 大学院・医学研究科, 助教授 (20142713)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2001: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2000: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | Bioluminescence / Transcription / Clock genes / Suprachiasmatic nucleus / Luciferase / perfusion culture / Multi-electric dish |
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| Research Abstract |
We developed a system in which multiple gene expressions in a single cell were recorded as bioluminescence of different wave lengths. The method is applied for real-time analyzes of molecules in living cells.
1. Development of a perfusion culture system : In order to monitor the expression of a particular gene of cultured cells, we developed a perfusion system in which reporter activity was continuously measured. In this study, we used a luciferase of a secretion type (vargula hilgendorfii), the DNA of which was connected with the downstream of growth hormone (GH) promoter. The amount of luciferase protein in the perfusion culture medium and bioluminescence showed a clear positive correlation and proved a reliability of bioluminescence measurement. We also examined the time course of synthesis, intracellular transport and secretion of reporter luciferase by using specific blockers for transcription, translation or release of secretion vesicles.
2. Construction of a clock gene monitoring
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vector : We constructed a vector in which a mPer1 promoter region of 8.1kb was connected to the upstream of firefly luciferase DNA. They were transfected to the NIH3T3 cell line and stable transformants were obtained. The reporter system was evaluated by adding CLOCK/BMAL1 expression vectors and observing the increase of bioluminescence.
3. Development of a luminescence detection apparatus with high sensitivity :
1) A novel method for detecting absolute levels of luminescence
(1) A factor for transformation of relative intensity of luminescence to an absolute value was obtained by laser.
(2) The standardized solution of luciferase was calibrated using an obtained transform factor.
(3) The unknown sample was quantified its reporter activity by the above mention standard curve.
2) Development of multi-color detection apparatus for living cells
We developed a detection system in which more than one bioluminescence of different wave lengths were monitored simultaneously using specific filters and intensive CCD camera. Less
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