Generation of genetically engineered hamsters for circadian studies

用于昼夜节律研究的基因工程仓鼠的产生

• 批准号: 9224469
• 负责人: ERIC L BITTMAN
• 金额: $ 19.66万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9224469
• 关键词: Affect; American; Animal Model; Animals; Arousal; Back; Behavior; Behavioral; Biological Rhythm; Brain; Cell physiology; Cells; Chronobiology; Circadian Rhythms; Code; Coupling; Development; Disease; Disease model; Ebola virus; Employee Strikes; Enzymes; Estrous Cycle; Event; Exhibits; Feedback; Fibroblasts; Future; Gene Expression; Gene Mutation; Generations; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Genomics; Hamster Cell Line; Hamsters; Health; Heart Diseases; Hypothalamic structure; Immune; Incidence; Jet Lag Syndrome; Lead; Light; Luciferases; Malignant Neoplasms; Mental disorders; Meridians; Mesocricetus auratus; Metabolic Diseases; Metabolism; Methodology; Methods; Molecular; Monitor; Mus; Mutation; Neurologic; Neurosecretory Systems; Obesity; Organ; Organism; Output; Pacemakers; Pathology; Peripheral; Phase; Photoperiod; Physiological; Physiology; Predisposition; Process; Property; Proteins; Reporter; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Role; Running; Schedule; Signal Transduction; Sleep; Speed; Study models; System; Technology; Therapeutic; Time; Transgenic Organisms; Transplantation; Transposase; Travel; abstracting; addiction; base; casein kinase; circadian pacemaker; disorder risk; feeding; gain of function; improved; mutant; nervous system disorder; neurogenesis; novel therapeutics; operation; research study; response; shift work; sleep syndrome; success; suprachiasmatic nucleus; tau Proteins; tau mutation; tool; tool development

Abstract The present research will explore and develop a transgenic approach to producing a bioluminescent reporter strain of Syrian hamster that will be a valuable tool for circadian research. The tau hamster was the first mammalian period mutant to be discovered. Transplantation experiments using tau hamsters established that suprachiasmatic nucleus (SCN) regulates the daily scheduling of physiology and behavior. Discovery that tau is a gain of function of casein kinase 1ε was critical to achieving an understanding of the operation of transcriptional-translational feedback loops that generate circadian rhythms, both in the brain's pacemaker and in peripheral oscillators. We recently discovered duper, a new mutation in hamsters which, like tau, speeds up the circadian clock. Duper is not a change in the coding region of casein kinase or any other known clock gene, and the mutation does not affect clock speed in fibroblasts. Thus duper is unlikely to affect the TTFL, but more probably alters coupling relationships within the SCN. Duper causes a striking reduction in jet lag. Our experiments will validate the reporter strain by comparing luminescent traces with results from qRT-PCR. We will then compare the impact of the tau and duper mutations on circadian rhythms in order to determine whether shortening of period through effects on the pacemaker vs alterations in the TTFL have different consequences for organismal function. Although hamsters have provided a valuable model for studies of biological rhythms, their potential as a genetic tool has yet to be realized. We have recently contributed to the first draft of the hamster genome and established methods for making transgenic hamsters. We have employed piggyBac methodology to insert circadian reporters into a hamster cell line. This technology will now enable us to study organismal function. In the first application of the reporter strain, we will determine effects of duper upon the core cell-autonomous feedback loops and upon the coordination of pacemaker function. This will shed light on the mechanisms of circadian desynchrony. Unlike the species in which transgenic reporter strains have thus far been produced, hamsters have a particularly regular, circadian-based estrous cycle and a strong photoperiodic response. Thus development of the hamster reporter strain will make possible future applications to investigate neuroendocrine function. Finally, the importance of the hamster as a disease model for several pathologies including MERS and Ebola, and the relevance of jet lag to the incidence of disease in an era of frequent trans-meridian travel, indicate that the reporter strain will be of widespread utility. Given the importance of circadian organization in behavior and physiology, this research will reveal mechanisms that underlie neurologic diseases, sleep deficiencies and metabolic disorders, and lead to development of new therapies.

摘要 本研究将探索和开发一种产生生物发光的转基因方法 叙利亚仓鼠的报告菌株，这将是一个有价值的工具，昼夜节律的研究。tau仓鼠是 第一个被发现的哺乳动物周期突变体使用tau仓鼠的移植实验 确定视交叉上核（SCN）调节生理的日常安排， 行为发现tau蛋白是酪蛋白激酶1ε功能的获得，这对于实现 理解产生昼夜节律的转录-翻译反馈回路的运作 节奏，无论是在大脑的起搏器和周边振荡器。我们最近发现了duper， 仓鼠的新突变，像tau一样，加快了生物钟。Duper不是一个变化， 酪蛋白激酶或任何其他已知的时钟基因的编码区，并且该突变不影响时钟 成纤维细胞中的速度。因此，duper不太可能影响TTFL，但更可能改变耦合 SCN内部的关系。杜珀能显著减少时差反应。我们的实验将验证 通过比较发光迹线与来自qRT-PCR的结果来鉴定报告菌株。我们将比较 tau和duper突变对昼夜节律的影响，以确定是否 通过对起搏器的影响缩短周期与TTFL的改变具有不同的 对生物功能的影响。 尽管仓鼠为研究生物节律提供了一个有价值的模型，但它们的潜力 作为一种遗传工具还有待实现。我们最近为仓鼠的初稿做出了贡献 基因组和建立的方法，使转基因仓鼠。我们雇佣了piggyBac 将昼夜节律报告基因插入仓鼠细胞系的方法。这项技术将使我们能够 研究有机体功能。在报告菌株的第一次应用中，我们将确定duper 核心细胞自主反馈回路和起搏器功能的协调。这 将揭示昼夜节律的机制。与转基因生物不同， 到目前为止，已经产生了报告菌株，仓鼠有一个特别有规律的，基于昼夜节律的 发情周期和强烈的光周期反应。因此，仓鼠报告菌株的开发将 使未来研究神经内分泌功能成为可能。最后， 仓鼠作为包括MERS和埃博拉在内的几种病理学的疾病模型， 在疾病发病滞后、跨经络旅行频繁的时代，表明记者应变 将具有广泛的实用性。鉴于昼夜节律在行为和生理学中的重要性， 这项研究将揭示神经系统疾病、睡眠不足和代谢紊乱的机制。 疾病，并导致新疗法的开发。