JoVE Journal > Biology
Summary
Abstract
Protocol
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Biologia

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

Published: September 27, 2012
doi:
10.3791/4234
, , , ,
1Department of Biological Sciences,The University of Memphis

Summary

生物钟单个细胞内发挥作用，也就是说，它们是细胞的自我。在这里，我们描述了使用非侵入性，实时荧光素酶为基础的生物发光技术产生的细胞自主的时钟模型的方法。记者细胞提供听话的，功能性的昼夜节律生物学研究的模型系统。

Abstract

在哺乳动物中，许多方面的行为和生理的，如睡眠-觉醒周期和肝脏代谢的调节内源性生物钟（审阅1,2）。时间的昼夜节律系统是一个分层的多振荡器网络，与中央的时钟位于视交叉上核（SCN）同步和协调其他额外的SCN和外设时钟1,2。单个细胞的生成和维护的昼夜节律3,4，这些振荡器在有机体份额显着相似的生化负反馈机制的不同组织类型的功能单元。然而，由于在神经元网络级在SCN和有机体一级通过节奏，系统性的线索的相互作用，在有机体一级的昼夜节律不一定细胞自主5-7。相对于传统的研究在体内的自发活动和SCN外植体离体 ，CELL-总部设在体外实验中可以发现的细胞自主的昼夜缺陷5,8。在战略上，基于细胞的实验模型更易于处理的表型特征和基本时钟机制的快速发现5,8-13。

因为昼夜节律是动态的，纵向的高时间分辨率的测量来评估时钟功能。近年来，实时使用萤火虫荧光素酶的生物发光记录，记者已成为一种常见的技术研究在哺乳动物中的昼夜节律14,15，因为它允许分子节奏的持久性和动态检查。细胞的自我监控的昼夜节律基因表达，荧光素酶记者可以被引入到细胞通过瞬时转染13,16,17或稳定转导5,10,18,19。在这里，我们描述了一个稳定传导协议的慢病毒介导的基因传递。 Ţ他慢病毒载体系统是优于传统的方法，如瞬时转染和生殖传输，因为它的效率和多功能性：它可以有效地提供稳定整合到宿主基因组的分裂和非分裂细胞20。记者细胞系建立之后，时钟功能的动态可以检查通过生物发光记录。我们首先介绍了新一代的P（Per2基因 ）-D 吕克记者的线条，然后和其他昼夜记者从目前的数据。在这些实验中，3T3小鼠成纤维细胞和U2OS人骨肉瘤细胞被用作细胞模型。我们还讨论了不同的方式使用这些时钟模型在昼夜节律的研究。这里描述的方法可以应用到多种类型的细胞，研究细胞和分子基础的生物钟，并在处理其他生物系统的问题可能会被证明是有用的。

Protocol

1。构建慢病毒荧光素酶记者哺乳动物的昼夜记者的结构通常包含一个表达盒，其中一昼夜的荧光素酶基因启动子的融合。双方结扎和重组基于策略通常用于DNA克隆。作为一个例子，在这里，我们描述了一个重组的Gateway克隆方法产生一个P（Per2基因 ）-D 吕克慢记者，在不稳定的荧光素酶 （D 吕克 ）的小鼠Per2基因启动子的控制下。 …

Discussion

1。目前的协议的修改

1.1记录装置和吞吐量的考虑

由于其商用中，LumiCycle（Actimetrics）已成为最常用的自动光度计设备实时记录4,5,9,19,29-31的 。 LumiCycle采用光电倍增管（PMT）作为光探测器，它提供了极高的灵敏度和低噪声14，因此特别适合于数据采集极其暗淡的基于荧光素酶发光。其他相似的PMT的设备（ 例如 ，克罗诺斯，阿托公司; POL…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作是支持的，部分由美国国家科学基金会（IOS-0920417）（ACL）。

Materials

Name of reagent Company Catalogue number Comments
DMEM HyClone SH30243FS For regular cell growth
DMEM Invitrogen 12100-046 For luminometry
FBS HyClone SH3091003  
Pen/Strep/Gln(100x) HyClone SV3008201  
B-27 Invitrogen 17504-044  
D-Luciferin Biosynth L-8220  
Poly-L-lysine Sigma P4707  
Polybrene Millipore TR-1003-G  
Forskolin Sigma F6886  
All other chemicals Sigma    
Equipment
Tissue culture incubator     5% CO2 at 37°C
Tissue culture hood     BSL-2 certified
Light & fluorescent microscope     Phase contrast optional
LumiCycle Actimetrics    
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Reppert, S. M., Weaver, D. R. Coordination of circadian timing in mammals. Nature. 418, 935-941 (2002).
  2. Hastings, M. H., Reddy, A. B., Maywood, E. S. A clockwork web: circadian timing in brain and periphery, in health and disease. Nat. Rev. Neurosci. 4, 649-661 (2003).
  3. Nagoshi, E. Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell. 119, 693-705 (2004).
  4. Welsh, D. K. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Curr. Biol. 14, 2289-2295 (2004).
  5. Liu, A. C. Intercellular coupling confers robustness against mutations in the SCN circadian clock network. Cell. 129, 605-616 (2007).
  6. Kornmann, B. System-driven and oscillator-dependent circadian transcription in mice with a conditionally active liver clock. PLoS Biol. 5, e34 (2007).
  7. Hogenesch, J. B., Herzog, E. D. Intracellular and intercellular processes determine robustness of the circadian clock. FEBS Lett. 585, 1427-1434 (2011).
  8. DeBruyne, J. P., Weaver, D. R., Reppert, S. M. Peripheral circadian oscillators require CLOCK. Curr. Biol. 17, 538-539 (2007).
  9. Liu, A. C. Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms. PLoS Genet. 4, e1000023 (2008).
  10. Zhang, E. E. A genome-wide RNAi screen for modifiers of the circadian clock in human cells. Cell. 139, 199-210 (2009).
  11. Baggs, J. E. Network features of the mammalian circadian clock. PLoS Biol. 7, e52 (2009).
  12. Hirota, T. High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIalpha as a clock regulatory kinase. PLoS Biol. 8, e1000559 (2010).
  13. Ukai-Tadenuma, M. Delay in feedback repression by cryptochrome 1 is required for circadian clock function. Cell. 144, 268-281 (2011).
  14. Yamazaki, S., Takahashi, J. S. Real-time luminescence reporting of circadian gene expression in mammals. Methods Enzymol. 393, 288-301 (2005).
  15. Welsh, D. K., Imaizumi, T., Kay, S. A. Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and mammalian cells. Methods Enzymol. 393, 269-288 (2005).
  16. Sato, T. K. Feedback repression is required for mammalian circadian clock function. Nat. Genet. 38, 312-319 (2006).
  17. Ueda, H. R. System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nat. Genet. 37, 187-192 (2005).
  18. Brown, S. A. The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol. 3, e338 (2005).
  19. Hirota, T. A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta. Proc. Natl. Acad. Sci. U.S.A. 105, 20746-20751 (2008).
  20. Tiscornia, G., Singer, O., Verma, I. M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241-245 (2006).
  21. Ueda, H. R. A transcription factor response element for gene expression during circadian night. Nature. 418, 534-539 (2002).
  22. Zufferey, R., Donello, J. E., Trono, D., Hope, T. J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886-2892 (1999).
  23. Buhr, E. D., Yoo, S. H., Takahashi, J. S. Temperature as a universal resetting cue for mammalian circadian oscillators. Science. 330, 379-385 (2010).
  24. Balsalobre, A., Damiola, F., Schibler, . U.A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell. 93, 929-937 (1998).
  25. Savelyev, S. A., Larsson, K. C., Johansson, A., Lundkvist, G. B. S. Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus. J. Vis. Exp. (48), e2439 (2011).
  26. Akashi, M., Ichise, T., Mamine, T., Takumi, T. Molecular mechanism of cell-autonomous circadian gene expression of Period2, a crucial regulator of the mammalian circadian clock. Mol. Biol. Cell. 17, 555-565 (2006).
  27. Ohno, T., Onishi, Y., Ishida, N. A novel E4BP4 element drives circadian expression of mPeriod2. Nucleic Acids Res. 35, 648-655 (2007).
  28. Maier, B. A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes Dev. 23, 708-718 (2009).
  29. Yoo, S. H. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 101, 5339-5346 (2004).
  30. Liu, A. C., Lewis, W. G., Kay, S. A. Mammalian circadian signaling networks and therapeutic targets. Nat. Chem. Biol. 3, 630-639 (2007).
  31. Ko, C. H. Emergence of noise-induced oscillations in the central circadian pacemaker. PLoS Biol. 8, e1000513 (2010).
  32. Izumo, M., Johnson, C. H., Yamazaki, S. Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: temperature compensation and damping. Proc. Natl. Acad. Sci. U.S.A. 100, 16089-16094 (2003).
  33. Izumo, M., Sato, T. R., Straume, M., Johnson, C. H. Quantitative analyses of circadian gene expression in mammalian cell cultures. PLoS Comput. Biol. 2, e136 (2006).
  34. Chen, Z. Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening. Proc. Natl. Acad. Sci. U.S.A. 109, 101-106 (2011).
  35. Yamaguchi, S. Synchronization of cellular clocks in the suprachiasmatic nucleus. Science. 302, 1408-1412 (2003).
  36. Akashi, M., Hayasaka, N., Yamazaki, S., Node, K. Mitogen-activated protein kinase is a functional component of the autonomous circadian system in the suprachiasmatic nucleus. J. Neurosci. 28, 4619-4623 (2008).
  37. Hoshino, H., Nakajima, Y., Ohmiya, Y. Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging. Nat. Methods. 4, 637-639 (2007).
  38. Asai, M. Visualization of mPer1 transcription in vitro: NMDA induces a rapid phase shift of mPer1 gene in cultured SCN. Curr. Biol. 11, 1524-1527 (2001).
  39. Wilsbacher, L. D. Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo. Proc. Natl. Acad. Sci. U.S.A. 99, 489-494 (2002).
  40. Yamazaki, S. Resetting central and peripheral circadian oscillators in transgenic rats. Science. 288, 682-685 (2000).
  41. Welsh, D. K., Noguchi, T., Yuste, R. Cellular bioluminescence imaging. Imaging: A Laboratory Manual. , 369-385 (2011).
  42. Nakajima, Y. Enhanced beetle luciferase for high-resolution bioluminescence imaging. PLoS One. 5, e10011 (2010).
  43. Guilding, C. A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus. Mol. Brain. 2, 28 (2009).
  44. O’Neill, J. S. cAMP-dependent signaling as a core component of the mammalian circadian pacemaker. Science. 320, 949-953 (2008).
  45. Fuller, P. M., Lu, J., Saper, C. B. Differential rescue of light- and food-entrainable circadian rhythms. Science. 320, 1074-1077 (2008).
  46. Mukherjee, S. Knockdown of Clock in the ventral tegmental area through RNA interference results in a mixed state of mania and depression-like behavior. Biol. Psychiatry. 68, 503-511 (2010).
  47. Saijo, K. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell. 137, 47-59 (2009).
  48. Elias, G. M. Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins. Neuron. 52, 307-320 (2006).
  49. Isojima, Y. CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock. Proc. Natl. Acad. Sci. U.S.A. 106, 15744-15749 (2009).
  50. Bucan, M., Abel, T. The mouse: genetics meets behaviour. Nat. Rev. Genet. 3, 114-123 (2002).
  51. Hughes, M. E. Harmonics of circadian gene transcription in mammals. PLoS Genet. 5, e1000442 (2009).
  52. Atwood, A. Cell-autonomous circadian clock of hepatocytes drives rhythms in transcription and polyamine synthesis. Proc. Natl. Acad. Sci. U.S.A. 108, 18560-18565 (2011).
  53. Panda, S. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 109, 307-320 (2002).

Tags

Cell-autonomous Circadian Clock RhythmsGene ExpressionLuciferase Bioluminescence ReportersMammalian Circadian ClocksSleep-wake CyclesLiver MetabolismEndogenous Circadian ClocksSuprachiasmatic Nucleus (SCN)Peripheral ClocksOscillatorsNegative Feedback MechanismCell-based In Vitro AssaysLocomotor ActivitySCN Explants Ex VivoCell-based ModelsClock FunctionTemporal ResolutionReal-time Bioluminescence RecordingFirefly Luciferase Reporter

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Ramanathan, C., Khan, S. K., Kathale, N. D., Xu, H., Liu, A. C. Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters. J. Vis. Exp. (67), e4234, doi:10.3791/4234 (2012).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image