2001 Fiscal Year Final Research Report Summary
Multichannel analysis and monitoring of multiple gene expressions by using bioluminescent reporters
| Project/Area Number |
12557005
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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 |
Environmental physiology (including Physical medicine and Nutritional physiology)
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
HONMA Sato Hokkaido Univ., Grad. School of Medicine, Associate Prof., 大学院・医学研究科, 助教授 (20142713)
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| Co-Investigator(Kenkyū-buntansha) |
OHMIYA Yoshihiro National Institute of AIST, Chief researcher, 産業技術総合研究所, 主任研究員 (20223951)
SHIRAKAWA Tetsuo Hokkaido Univ., Grad. School of Dentistry, Lecturer, 大学院・歯学研究科附属病院, 講師 (00187527)
ABE Hiroshi Hokkaido Univ., Grad. School of Medicine, Lecturer, 大学院・医学研究科, 講師 (80201896)
KUBOTA Hidehiro Atto Coorperation, Research Manager, 技術開発部・学術研究課, 課長(研究職)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Bioluminescence / Multi-reporter system / Transfection / Luciferase / Transcription factor |
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| Research Abstract |
By transfecting reporter genes which encode luciferase emitting light with different wave lengths, we monitored expression of multiple genes in single cells. The method was applied for two dimensional and time-course analyzes to visualize the interactions of molecules within living cells.
(1) Cloning of novel bioluminescence reporter genes and construction of multi-color reporter vector : Two luciferases of the railroad-worm emit red and green lights by catalyzing the same substrate. The dual reporter vector was constructed by inserting the cDNAs, one under the vasopressin and the other under CMV promoter. Although the light emission could be detected, the luminescence in the mammalian cell was weak. Therefore, dual reporter with higher intensity was constructed using vargula hilgendorfii luciferase and firefly luciferase. Promoter sequence of growth hormone (GH) gene was connected to the upstream of vargula luciferase cDNA and the reporter vector was transfected to the GH3 cell. The st
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able colony of GH3 cells was further transfected firefly luciferase. Luminescence from vargula and firefly luciferases was separately monitored from GH3 cells continuously.
(2) Construction of multi-color monitoring system : We constructed the detection system consisting of dark box kept 37 ℃ in which a turn table carrying 6 grass-bottom petri dishes rotates on the cooled CCDcamera. A luminocapter counted the photons after separating those with the peak wavelength through liquid crystal filters.
(3) Cloning of promoter sequences and continuous monitoring of targeted gene expression in living cells : Transcriptional regulation was analyzed using various length of promoter sequences of mouse Per1 and BMAL1 genes connected with either GFP or firefly luciferase cDNA. We identify the regulatory region in the promoter and constructed dual color reporter to monitor Per1 and BMAL1 expressions. We are constructing the transgenic mice line to study the molecular clock mechanisms monitoring the expression of the two clock genes. Less
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