1996 Fiscal Year Annual Research Report

生物発光を用いた遺伝子発現のリアルタイム・モニタリングシステムの開発

Research Project

Project/Area Number	06557005
Research Institution	Hokkaido University
Principal Investigator	本間研一北海道大学, 医学部, 教授 (40113625)
Co-Investigator(Kenkyū-buntansha)	石浦正寛名古屋大学, 理学部, 助教授 (20132730) 近藤孝男名古屋大学, 理学部, 教授 (10124223) 安倍博北海道大学, 医学部, 助手 (80201896) 勝野由美子北海道大学, 医学部, 助手 (80177419)
Keywords	Aequorin / 発光蛋白質 / Caイオン / トランスジェニックマウス / NIH3T3 / レポーター遺伝子 / 遺伝子発現 / リアルタイム計測
Research Abstract	1.Aequorin発光系を組み込んだトランスジェニックマウスにおける遺伝子発現のモニタリング発光蛋白質であるaequorinの発現を高感度CCDカメラを用いて光学的に測定し、遺伝子発現を細胞内Caイオン濃度を操作することで観察した。神経特異性エノラーゼのプロモータ領域下流にaequorin遺伝子を組み込んだトランスジェニックマウスの脳を試料とした。リコンビナントapoaequorinがCaイオンによって容量依存的に発光することを確かめたのち、aequorinおよび神経特異エノラーゼ遺伝子の挿入をPRC法にて確認したマウスの脳組織を大脳、小脳、視交叉上核を含む視床下部に分けてホモジェネイトした。基質であるセレンテラジンを加えてインキュベイトしたのち、自作の光子量測定装置を用いて暗黒環境下遠隔操作でCaイオンを試料に滴下した。その結果、大脳皮質において有意な発光がみられ、遺伝子発現の光学的観察に成功した。 2.NIH3T3細胞をもちいた生物発光系の確立生きた細胞で遺伝子発現をモニターする目的で、NIH3T3細胞にaequorin遺伝子を導入し、遺伝子発現を細胞内Caイオン濃度の操作で継時的に観察した。CMVプロモーターの下流領域にaequorin遺伝子を挿入したベクター(pRK7)を作成し、NIH3T3細胞にトランスフェクトした。遺伝子導入36時間後にCaイオンを投与し、生物発光を定量して遺伝子発現をモニターした。その結果、Caイオンによる発光量は容量依存的に増加し、トランスフェクトされた遺伝子が発現されていることが確認された。さらにKC1を用いて細胞を脱分極させたところ、発光量が瞬時に増加し、発光量の増加は40秒ほど持続した。以上の結果から、生きた細胞でaequorin遺伝子の発現が確認でき、生物発光レポーター遺伝子を用いる遺伝子発現のリアルタイム計測が可能となった。

Research Products

(15 results)

All Other

All Publications (15 results)

[Publications] Hashimoto, S.: "Midday exposure to bright light changes the circadian organization of plasma melatonin rhythm in humans." Neurosci. Lett.(印刷中). (1997)
[Publications] Hashimoto, S.: "Free-running circadian rhythm of melatonin in a sighted young man despite a 24-hr sleep pattern : a non-24-hour circadian syndrome." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Nakamura, K.: "A sighted man with non-24-hour sleep-syndrome showed damped plasma melatonin rhythm." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Nakamura, K.: "Daily melatonin intake resets circadian rhythms of a sighted man with no-24-hour sleep-wake syndrome who lacks nocturnal melatonin rise." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Honma, K.: "Sleep and melatonin rhythm in humans." Sleep and Sleep Disorders - from Molecule to Behavior. (印刷中). (1997)
[Publications] Hashimoto, S.: "Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans." Neruscience Letters. 220. 129-132 (1996)
[Publications] Honma, S.: "Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continous light in the rat." Neruscience Letters. 216. 49-52 (1996)
[Publications] Honma, S.: "Aging affects development and persistence of feeding assoicated circadian rhythm in rat plasma corticosterone." American Journal of Physiology. 271. R1514-R1520 (1996)
[Publications] Honma, S.: "Circadian rhythm and response to light of extracellular glutamate and aspartate in rat suprachiasmativc nucleus." American Journal of Physiology. 271. R579-R585 (1996)
[Publications] Yoshihara, T.: "Dissociationm of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation." American Journal of Physiology. 271. E239-E245 (1996)
[Publications] Yoshihara, T.: "Prefeeding release of paraventricular neuropeptide Y is mediated by the ascending noradrenergic neurons in rats." American Journal of Physiology. 270. E596-E600 (1996)
[Publications] Yoshihara, T.: "Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus." American Journal of Physiology. 270. E589-E595 (1996)
[Publications] Hashimoto, S.: "Melatonin rhythm is not shifted by lights which suppress noctural melatonin in humans under entrainment." American Journal of Physiology. 270. R1073-R1077 (1996)
[Publications] Abe. H.: "Substance P receptor regulates the photic induction of Fos-like prontein in the suprachiasmatic nucleus of Syrian hamsters." Brain Research. 708. 135-142 (1996)
[Publications] Yoshihara, T.: "Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats." Brain Research. 707. 119-121 (1996)

1996 Fiscal Year Annual Research Report

生物発光を用いた遺伝子発現のリアルタイム・モニタリングシステムの開発

Principal Investigator

本間 研一 北海道大学, 医学部, 教授 (40113625)

Research Products

[Publications] Hashimoto, S.: "Midday exposure to bright light changes the circadian organization of plasma melatonin rhythm in humans." Neurosci. Lett.(印刷中). (1997)

[Publications] Hashimoto, S.: "Free-running circadian rhythm of melatonin in a sighted young man despite a 24-hr sleep pattern : a non-24-hour circadian syndrome." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Nakamura, K.: "A sighted man with non-24-hour sleep-syndrome showed damped plasma melatonin rhythm." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Nakamura, K.: "Daily melatonin intake resets circadian rhythms of a sighted man with no-24-hour sleep-wake syndrome who lacks nocturnal melatonin rise." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Honma, K.: "Sleep and melatonin rhythm in humans." Sleep and Sleep Disorders - from Molecule to Behavior. (印刷中). (1997)

[Publications] Hashimoto, S.: "Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans." Neruscience Letters. 220. 129-132 (1996)

[Publications] Honma, S.: "Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continous light in the rat." Neruscience Letters. 216. 49-52 (1996)

[Publications] Honma, S.: "Aging affects development and persistence of feeding assoicated circadian rhythm in rat plasma corticosterone." American Journal of Physiology. 271. R1514-R1520 (1996)

[Publications] Honma, S.: "Circadian rhythm and response to light of extracellular glutamate and aspartate in rat suprachiasmativc nucleus." American Journal of Physiology. 271. R579-R585 (1996)

[Publications] Yoshihara, T.: "Dissociationm of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation." American Journal of Physiology. 271. E239-E245 (1996)

[Publications] Yoshihara, T.: "Prefeeding release of paraventricular neuropeptide Y is mediated by the ascending noradrenergic neurons in rats." American Journal of Physiology. 270. E596-E600 (1996)

[Publications] Yoshihara, T.: "Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus." American Journal of Physiology. 270. E589-E595 (1996)

[Publications] Hashimoto, S.: "Melatonin rhythm is not shifted by lights which suppress noctural melatonin in humans under entrainment." American Journal of Physiology. 270. R1073-R1077 (1996)

[Publications] Abe. H.: "Substance P receptor regulates the photic induction of Fos-like prontein in the suprachiasmatic nucleus of Syrian hamsters." Brain Research. 708. 135-142 (1996)

[Publications] Yoshihara, T.: "Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats." Brain Research. 707. 119-121 (1996)

本間研一北海道大学, 医学部, 教授 (40113625)