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Development of multidimensional real-time monitoring system for neuronal activity by plural bioluminescent reporters

Research Project

Project/Area Number 12558091
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Neuroscience in general
Research InstitutionHOKKAIDO UNIVERSITY

Principal Investigator

HONMA Ken-ichi  Hokkaido Univ., Grad. School of Medicine, Prof., 大学院・医学研究科, 教授 (40113625)

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)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
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)
KeywordsBioluminescence / Transcription / Clock genes / Suprachiasmatic nucleus / Luciferase / perfusion culture / Multi-electric dish
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 … More 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

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (44 results)

All Other

All Publications (44 results)

  • [Publications] Shirakawa, T.: "Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons"Eur. J. Neurosci.. 12. 2833-2838 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinohara, K.: "Circadian release of excitatory amino acids in the suprachiasmatic nucleus culture is Ca^<2+>-independent"Neurosci. Res.. 36. 245-250 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ikeda, M.: "cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2 : Its mRNA expression, subcellular distribution, and chromsomal localization"Biochem. Biophys. Res. Commun.. 275. 493-502 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Masubuchi, S.: "Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats"Eur. J. Neuroscience. 12. 4206-4214 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Honma, S: "Synaptic communication of cellular oscillations in the rat suprachiasmatic neurons"Neurosci. Lett.. 294. 113-116 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tanahashi, Y: "Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase"Analylica Biochemistry. 289. 260-266 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Namihira, M: "Circadian pattern, light responsiveness and localization of rPer1 and rPer2 gene expression in the rat retina"Neuroreport. 12. 471-475 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Abe, H.: "Clock gene expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice"Mol. Brain Res.. 87. 92-99 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki, T.: "Quantitative trait locus analysis of abnormal circadian period in CS mice"Mammalian Genome. 12. 272-277 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shirakawa, T.: "Multiple oscillators in the suprachiasmatic nucleus"Chronobiol. Int.. 18. 371-387 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakamura, W.: "Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus"Eur. J. Neurosci.. 14. 666-674 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yoshihara, T.: "Liquid mean attenuates meal anticipation in rat adrenocortical activity"Physiol. Behav.. 74. 133-137 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Masubuchi, S.: "Circadian activity rhythm in Clock mutant mouse by methamphetamine"Eur. J. Neurosc.. 14. 1177-1180 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Abe, H.: "Behavioral rhythm splitting in the SC mouse is related to clock gene expressions outside the suprachiasmatic nucleus"Eur. J. Neurosci.. 14. 1121-1128 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Honma, S.: "Zeigebers, Entrainment and Masking of the Circadian System"Hokkaido Univ. Press. 299 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shirakawa, T.: "Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons"Eur. J. Neurosci.. 12. 2833-2838 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinohara, K.: "Circadian release of excitatory amino acids in the suprachiasmatic nucleus culture is Ca^<2+> - independent"Neurosci. Res.. 36. 245-250 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ikeda, M.: "cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2 : Its mRNA expression, subcellular distribution, and chromosomal localization"Biochem. Biophys. Res. Commun.. 275. 493-502 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Masubuchi, S.: "Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats"Eur. J. Neuroscience.. 12. 4206-4214 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Honma, S.: "Synaptic communication of cellular oscillations in the rat suprachiasmatic neurons"Neurosci. Lett.. 294. 113-116 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tanahashi, Y.: "Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase"Analytica Biochemistry. 289. 260-266 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Namihira, M.: "Circadian pattern, light responsiveness and localization of rPer1 and rPer2 gene expression in the rat retina"Neuroreport. 12. 471-475 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Abe, H.: "Clock gene expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice"Mol. Brain Res.. 87. 92-99 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki, T.: "Quantitative trait locus analysis of abnormal circadian period in CS mice"Mammalian Genome. 12. 272-277 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shirakawa, T.: "Multiple oscillators in the suprachiasmatic nucleus"Chronobiol. Int.. 18. 371-387 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakamura, W.: "Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus"Eur. J. Neurosci.. 14. 666-674 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yoshihara, T.: "Liquid mean attenuates meal anticipation in rat adrenocortical activity"Physiol. Behav.. 74. 133-137 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Masubuchi, S.: "Circadian activity rhythm in Clock mutant mouse by methamphetamine"Eur. J. Neurosc.. 14. 1177-1180 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Abe, H.: "Behavioral rhythm splitting in the SC mouse is related to clock gene expressions outside the suprachiasmatic nucleus"Eur. J. Neurosci.. 14. 1121-1128 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Honma, S.: "Zeigebers, Entrainment and Masking of the Circadian System"Hokkaido Univ. Press, Sapporo. 299 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Y.Tanahashi, et al.: "Continuous measurement of targeted promotor activity by a secreted bioluminescence reporter, Vargula hilgendorfii, luciferase"Anal Biochem. 289. 260-266 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] M.Namihira, et al.: "Circadian pattern, light responsiveness and localization of clock gene, rPer1 and rPer2, expressions in the rat retina"NeuroReport. 12. 471-475 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] H.Abe, et al.: "Clock genes expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice"Mol. Brain Res.. 87. 92-99 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] W.Nakamura, et al.: "Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus"Europ. J. Neurosci.. 14. 666-674 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] S.Masubuchi, et al.: "Circadian activity rhythm in methamphetamine-treated clock mutant mouse"Europ. J. Neurosci.. 14. 1177-1180 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Shirakawa, et al.: "Multiple oscillators in the suprachiasmatic nucleus"Chronobiol. Int.. 18. 371-387 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] K.Honma, S.Honma: "Zeitgebers, Entrainment and Masking of the Circadian System"Hokkaido University Press. 1-299 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] M.Ikeda, et al.: "cDNA Cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2 : Its mRNA expression, subcellular distribution, and chromosomal localization."Biochem.Biophys.Res.Comm.. 261. 493-502 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Shirakawa,et al.: "Synchronization of circadian firing rhythms in cultured rat suprachiasmatic nucleus."Europ.J.Neurosci.. 12. 2833-2838 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Masubuchi, et al.: "Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats."Europ.J.Neurosci.. 12. 4206-4214 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Namihira, et al.: "Circadian pattern, light responsiveness and localization of clock gene, rPer1 and rPer2, expressions in the rat retina."NeuroReport,. 12. 471-475 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Honma, et al.: "Synaptic communication of cellular oscillations in the rat suprachiasmatic neurons."Neurosci.Lett.. 294. 113-116 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] H.Abe, et al.: "Clock genes expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice."Mol.Brain Res.. (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Tanahashi, et al.: "Continuous measurement of targeted promotor activity by a secreted bioluminescence reporter, Vargula hilgendorfii, luciferase."Anal Biochem,. (2001)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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