Generation of genetically engineered hamsters for circadian studies

摘要 本研究将探索和开发一种转基因方法来生产生物发光 报道说，叙利亚仓鼠的品系将成为昼夜节律研究的宝贵工具。陶氏仓鼠是 发现了第一个哺乳动物时期的突变基因。利用tau仓鼠进行的移植实验 证实视交叉上核(SCN)调节日常的生理和 行为。发现tau是酪蛋白激酶1ε功能的获得是实现 理解产生昼夜节律的转录-翻译反馈环的运作 节律，在大脑的起搏器和外周振荡器中。我们最近发现了Duper，一种 仓鼠的新突变，像tau一样，加快了生物钟。DUPER并不是对 酪蛋白激酶编码区或任何其他已知的时钟基因，并且该突变不影响时钟 成纤维细胞的速度。因此，DUPER不太可能影响TTFL，但更有可能改变耦合 SCN内部的关系。DUPER能显著减少时差反应。我们的实验将验证 通过将荧光示踪与qRT-PCR结果进行比较，筛选出报告基因。然后我们将比较 Tau和duper突变对昼夜节律的影响 间期缩短对起搏器的影响与TTFL的改变有不同 对机体功能的影响。 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没有实现。我们最近为仓鼠的初稿做了贡献 基因组和制造转基因仓鼠的既定方法。我们已经使用了PiggyBac 将昼夜节律记者插入仓鼠细胞系的方法学。这项技术现在将使我们能够 研究人体机能。在报告菌株的第一次应用中，我们将确定DUPER的效果 根据核心细胞自主反馈环和起搏器功能的协调。这 将阐明昼夜节律去同步化的机制。不同于转基因的物种 到目前为止已经产生了报告菌株，仓鼠有特别规律的、以昼夜节律为基础的 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 使未来有可能应用于研究神经内分泌功能。最后，重要的是 仓鼠作为包括MERS和埃博拉在内的几种病理的疾病模型，以及JET的相关性 滞后于疾病发病率在一个经络旅行频繁的时代，表明记者的菌株 将具有广泛的实用价值。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机制。 疾病，并导致新疗法的发展。

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.