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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的目前的研究将探索和开发一种转基因方法来生产生物发光叙利亚仓鼠的报告品系将成为昼夜节律研究的宝贵工具。发现的第一个哺乳动物周期突变体使用 tau 仓鼠进行移植实验。确定视交叉上核 (SCN) 调节生理的日常安排 tau 是酪蛋白激酶 1ε 功能增强的发现对于实现这一目标至关重要。了解产生昼夜节律的转录翻译反馈环的运作我们最近发现了 duper，一种大脑起搏器和外周振荡器的节律。仓鼠的新突变与 tau 蛋白一样，加快了生物钟，这并不是生物钟的变化。酪蛋白激酶或任何其他已知时钟基因的编码区，并且突变不影响时钟因此，duper 不太可能影响 TTFL，但更有可能改变耦合。 SCN 内的 Duper 关系会显着减少时差反应，我们的实验将对此进行验证。然后我们将通过将发光迹线与 qRT-PCR 结果进行比较来确定报告菌株。 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而缩短周期与改变 TTFL 具有不同的效果对生物功能的影响。尽管仓鼠为生物节律研究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尚未实现，我们最近为仓鼠的初稿做出了贡献。基因组和建立转基因仓鼠的方法我们已经采用了piggyBac。将昼夜节律产生器插入仓鼠细胞系的方法现在使我们能够在报告菌株的首次应用中，我们将确定 duper 的生物学功能。取决于核心细胞自主反馈回路和起搏器功能的协调。将揭示与转基因物种不同的昼夜节律不同步机制。迄今为止，已经产生了报告菌株，仓鼠具有特别规律的、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品系的发育。使未来的应用成为可能，以研究神经内分泌功能。仓鼠作为多种疾病的疾病模型，包括中东呼吸综合征和埃博拉病毒，以及喷射的相关性在频繁跨经域旅行的时代，疾病的发生率存在滞后性，这表明报告者菌株鉴于昼夜节律组织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、睡眠不足和新陈代谢的潜在机制疾病，并导致新疗法的开发。