体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测
Summary
这种新开发的荧光技术能够长期监测自由运动小鼠视交叉上核 (SCN) 中昼夜节律基因的转录。
Abstract
该技术将光纤介导荧光记录与重组腺相关病毒基因记者的精确交付结合在一起。这种新的和易于使用的体内荧光监测系统, 以记录的转录节律的时钟基因, Cry1, 在视交叉上核 (SCN) 的自由移动小鼠。为此, Cry1转录荧光报告器被设计和包装成腺相关病毒。纯化, 浓缩病毒注射到小鼠的 SCN, 然后插入一根光纤, 然后固定在大脑的表面上。这些动物被送回自己的笼子, 并允许1月的手术后恢复期, 以确保足够的记者表达。荧光然后记录在自由移动的小鼠通过体内监测系统, 在我们的机构建设。在体内记录系统中, 488 nm 激光与 1 x 4 光束分配器耦合, 将光分成四个同等功率的激光励磁输出。这个设置使我们能够同时记录四动物。每个发射的荧光信号都是通过光电倍增管和数据采集卡收集的。与先前的活体生物钟记录技术的生物发光相比, 这种荧光体内记录系统允许在光周期中记录生物钟基因的表达。
Introduction
在哺乳动物中, 视交叉上核 (SCN) 支配着整个身体的昼夜节律, 以协调个体对外部环境变化的反应 (如光照、温度、压力等)1。核心时钟组件由Per1-3、 Cry1-2、时钟和Bmal1组成, 在调节每个细胞的昼夜节律时起着中枢作用。每个细胞在 SCN 包含转录激活剂, CLOCK/BMAL1, 作为一个 heterodimer, 以诱导表达的每和哭。每/哭复合, 然后抑制 CLOCK/BMAL1 的功能, 形成一个转录转换反馈回路, 约24小时完成2,3。
以往对 scn 的研究主要采用了前体4、5、6的体外营养切片培养方法, 虽然这种方法提供了有价值的信息, 但它的局限性抑制了我们获取有关其他脑核对 SCN 的影响的数据, 以及外源性刺激 (如光照) 对居住在这个关键区域的细胞的影响。在 2001年, 仁冈村的组是第一个使用生物发光系统在体内监测昼夜节律基因表达的 SCN 在自由移动小鼠7。肯-本间的小组在过去几年里进一步开发了生物荧光在体内记录系统在 SCN8,9,10。这些研究共同为研究人员提供了在恒定的黑暗或光脉冲后监测生物钟的能力。然而, 由于生物发光太暗, 无法在光/暗循环期间进行连续监测, 加上光是11昼夜节律性时钟所需的主要信号, 因此有不断增加的需求, 开发的实验方法, 克服了与生物发光记录相关的限制。本报告描述了一种基于荧光的系统, 用于监测自由移动小鼠体内的 SCN 生物钟。这种易于使用的方法允许在光/暗循环期间进行连续监测, 并允许在实时和高时间分辨率下长期观察 SCN 中生物钟基因的转录。
Protocol
Representative Results
Discussion
与前体方法相比, 如切片培养4,5, rt-pcr16,原位杂交17, 这要求动物被杀死,体内记录方法允许研究者们研究活着的动物的昼夜节律基因表达。因此, 这种技术提供了评估不同物理扰动 (如睡眠剥夺、压力、食物摄入等) 对神经昼夜节律的影响的能力。与体内生物发光7</su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢张实验室的各位成员提供了激动人心的讨论, 以及站内实验室的成员提供技术援助。这项研究由中国国家自然科学基金、2012CB837700 (E.E.Z. 和霍山) 资助的 31500860 (到霍山) 的973个项目中的 m.o.s. T, 并由北京市政府提供资金支持。E.E.Z. 得到中国 “全球青年专家招募计划” 的支持。
Materials
| KOD Plus Neo | TOYOBO | KOD-401 | Reagent |
| pVENUS-N1 | addgene | #61854 | Plasmid |
| pcDNA3.3_d2eGFP | addgene | #26821 | Plasmid |
| pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA | addgene | #20297 | Plasmid |
| MluI | Thermo Scientific | FD0564 | Reagent |
| EcoRI | Thermo Scientific | FD0274 | Reagent |
| Gibson Assembly Mix | NEB | E2611s | Reagent |
| Lipofectamine 2000 | Thermo Scientific | 12566014 | Reagent |
| Syringe Filter | EMD Millipore | SLHV033RS | 0.45 µm |
| HiTrap heparin columns | gelifesciences | 17-0406-01 | 1 mL |
| Amicon ultra-4 centrifugal filter | EMD Millipore | UFC810024 | 100,000 MWCO |
| Benzonase nuclease | Sigma-Aldrich | E1014 | Reagent |
| Sodium deoxycholate | Sigma-Aldrich | D5670 | Make fresh solution for each batch |
| mouse stereotaxic apparatus | B&E TEKSYSTEMS LTD | #SR-5M/6M | Equipment |
| pentobarbital | SigmaAldrich | #1507002 | Reagent |
| mouse stereotaxic apparatus | B&E TEKSYSTEMS LTD | #SR-5M/6M | Equipment |
| Hydrogen peroxide solution | SigmaAldrich | #216763 | Reagent |
| Optical Fiber | Thorlabs | FT200EMT | 0.39 NA, Ø200 µm |
| microsyringe pump | Nanoliter 2000 Injector, WPI | Equipment | |
| ceramic ferrule | Shanghai Fiblaser | 230 μm I.D., 2.5 mm O.D. | |
| Gene Observer | BiolinkOptics | Equipment |
References
- Welsh, D. K., Takahashi, J. S., Kay, S. A. Suprachiasmatic nucleus: cell autonomy and network properties. Annual Review of Physiology. 72, 551-577 (2010).
- Zhang, E. E., Kay, S. A. Clocks not winding down: unravelling circadian networks. Nature Reviews Molecular Cell Biology. 11, 764-776 (2010).
- Takahashi, J. S. Transcriptional architecture of the mammalian circadian clock. Nature Reviews Genetics. 18, 164-179 (2017).
- Yoo, S. H., et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proceedings of the National Academy of Sciences U S A. 101, 5339-5346 (2004).
- Davidson, A. J., Castanon-Cervantes, O., Leise, T. L., Molyneux, P. C., Harrington, M. E. Visualizing jet lag in the mouse suprachiasmatic nucleus and peripheral circadian timing system. European Journal of Neuroscience. 29, 171-180 (2009).
- Savelyev, S. A., Larsson, K. C., Johansson, A. S., Lundkvist, G. B. Slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus. Journal of Visualized Experiments. (48), (2011).
- Yamaguchi, S., et al. Gene expression: View of a mouse clock gene ticking. Nature. 409, 684-684 (2001).
- Ono, D., Honma, K. I., Honma, S. Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice. Science Reports. 5, 12310 (2015).
- Ono, D., Honma, S., Honma, K. Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms. European Journal of Neuroscience. 42, 3128-3137 (2015).
- Ono, D., et al. Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs. Proceedings of the National Academy of Sciences U S A. 114, E3699-E3708 (2017).
- Reppert, S. M., Weaver, D. R. Coordination of circadian timing in mammals. Nature. 418, 935-941 (2002).
- Liu, A. C., et al. Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms. PLoS Genetics. 4, e1000023 (2008).
- Maywood, E. S., et al. Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse. Proceedings of the National Academy of Sciences U S A. 110, 9547-9552 (2013).
- Ukai-Tadenuma, M., et al. Delay in feedback repression by cryptochrome 1 is required for circadian clock function. Cell. 144, 268-281 (2011).
- McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. J. Production and titering of recombinant adeno-associated viral vectors. Journal of Visualized Experiments. 57, e3348 (2011).
- Yamaguchi, Y., et al. Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag. Science. 342, 85-90 (2013).
- Nagano, M., et al. An abrupt shift in the day/night cycle causes desynchrony in the mammalian circadian center. Journal of Neuroscience. 23, 6141-6151 (2003).
- Golombek, D. A., Rosenstein, R. E. Physiology of Circadian Entrainment. Physiological Reviews. 90, 1063-1102 (2010).
- Mei, L., et al. Long-term in vivo recording of circadian rhythms in brains of freely moving mice. Proceedings of the National Academy of Sciences. 115, 4276-4281 (2018).
