Name: in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters
Uploaded: 2018-07-04T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

Vi takke medlemmerne i Zhang lab for at give stimulerende diskussioner og medlemmer i Zhan laboratoriet for teknisk bistand. Denne forskning blev støttet af tilskud 31500860 (til C.Z.) af NSFC, 2012CB837700 (til E.E.Z. og C.Z.) af programmet 973 fra M.O.S.T. i Kina, og af midler fra Beijing Municipal regering. E.E.Z. blev støttet af kinesiske "Rekruttering Program af Global Ungdom eksperter".

Alle procedurer i denne protokol blev gennemført med godkendelse af institutionelle Animal Care og brug udvalg (IACUC) af det nationale Institut for biologiske videnskaber, Beijing, i overensstemmelse med statslige bestemmelser i Kina.
1. opførelse af Cry1 fluorescens Reporter
Bemærk: Tidligere døgnrytmen undersøgelser ved hjælp af bioluminescens system2,12,13 ...

<ol>
	<li>Welsh, D. K., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suprachiasmatic+nucleus:+cell+autonomy+and+network+properties.">Suprachiasmatic nucleus: cell autonomy and network properties.</a> Annual Review of Physiology. 72, 551-577 (2010).</li><li>Zhang, E. E., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clocks+not+winding+down:+unravelling+circadian+networks.">Clocks not winding down: unravelling circadian networks.</a> Nature Reviews Molecular Cell Biology. 11, 764-776 (2010).</li><li>Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+architecture+of+the+mammalian+circadian+clock.">Transcriptional architecture of the mammalian circadian clock.</a> Nature Reviews Genetics. 18, 164-179 (2017).</li><li>Yoo, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proceedings of the National Academy of Sciences U S A. 101, 5339-5346 (2004).</li><li>Davidson, A. J., Castanon-Cervantes, O., Leise, T. L., Molyneux, P. C., Harrington, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+jet+lag+in+the+mouse+suprachiasmatic+nucleus+and+peripheral+circadian+timing+system.">Visualizing jet lag in the mouse suprachiasmatic nucleus and peripheral circadian timing system.</a> European Journal of Neuroscience. 29, 171-180 (2009).</li><li>Savelyev, S. A., Larsson, K. C., Johansson, A. S., Lundkvist, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+preparation,+organotypic+tissue+culturing+and+luciferase+recording+of+clock+gene+activity+in+the+suprachiasmatic+nucleus.">Slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Yamaguchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression:+View+of+a+mouse+clock+gene+ticking.">Gene expression: View of a mouse clock gene ticking.</a> Nature. 409, 684-684 (2001).</li><li>Ono, D., Honma, K. I., Honma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+ultradian+rhythms+of+clock+gene+expression+in+the+suprachiasmatic+nucleus+of+freely+moving+mice.">Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice.</a> Science Reports. 5, 12310 (2015).</li><li>Ono, D., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+PER2::LUC+rhythms+in+the+olfactory+bulb+of+freely+moving+mice+depend+on+the+suprachiasmatic+nucleus+but+not+on+behaviour+rhythms.">Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms.</a> European Journal of Neuroscience. 42, 3128-3137 (2015).</li><li>Ono, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+Per1+and+Bmal1+circadian+rhythms+in+the+suprachiasmatic+nucleus+in+parallel+with+behavioral+outputs.">Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs.</a> Proceedings of the National Academy of Sciences U S A. 114, E3699-E3708 (2017).</li><li>Reppert, S. M., Weaver, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+circadian+timing+in+mammals.">Coordination of circadian timing in mammals.</a> Nature. 418, 935-941 (2002).</li><li>Liu, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundant+function+of+REV-ERBalpha+and+beta+and+non-essential+role+for+Bmal1+cycling+in+transcriptional+regulation+of+intracellular+circadian+rhythms.">Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.</a> PLoS Genetics. 4, e1000023 (2008).</li><li>Maywood, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+core+circadian+feedback+loop+in+suprachiasmatic+nucleus+of+mCry1-luc+transgenic+reporter+mouse.">Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.</a> Proceedings of the National Academy of Sciences U S A. 110, 9547-9552 (2013).</li><li>Ukai-Tadenuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+in+feedback+repression+by+cryptochrome+1+is+required+for+circadian+clock+function.">Delay in feedback repression by cryptochrome 1 is required for circadian clock function.</a> Cell. 144, 268-281 (2011).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+titering+of+recombinant+adeno-associated+viral+vectors.">Production and titering of recombinant adeno-associated viral vectors.</a> Journal of Visualized Experiments. 57, e3348 (2011).</li><li>Yamaguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+genetically+deficient+in+vasopressin+V1a+and+V1b+receptors+are+resistant+to+jet+lag.">Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag.</a> Science. 342, 85-90 (2013).</li><li>Nagano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abrupt+shift+in+the+day/night+cycle+causes+desynchrony+in+the+mammalian+circadian+center.">An abrupt shift in the day/night cycle causes desynchrony in the mammalian circadian center.</a> Journal of Neuroscience. 23, 6141-6151 (2003).</li><li>Golombek, D. A., Rosenstein, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Circadian+Entrainment.">Physiology of Circadian Entrainment.</a> Physiological Reviews. 90, 1063-1102 (2010).</li><li>Mei, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+recording+of+circadian+rhythms+in+brains+of+freely+moving+mice.">Long-term in vivo recording of circadian rhythms in brains of freely moving mice.</a> Proceedings of the National Academy of Sciences. 115, 4276-4281 (2018).</li></ol>

I modsætning til ex vivo metoder, såsom skive kultur4,5, RT-PCR16, og i situ hybridisering17, der kræver, at dyrene aflives, den i vivo optagelse metode giver mulighed for efterforskere at studere døgnrytmen genekspression i et levende dyr. Som sådan, giver denne teknologi mulighed for at evaluere effekten af forskellige fysiske forstyrrelser (fx, søvn, stress, fødeindtagel...

I pattedyr styrer suprachiasmatic kernen (SCN) hele kroppens døgnrytme for at koordinere den enkeltes reaktion på eksogene miljømæssige ændringer (f.eks., lys, temperatur, stress, osv.)1. Core clock komponenter består af Per1-3, Cry1-2, urog Bmal1, og spiller en central rolle i reguleringen af døgnrytmen uret i hver celle. Hver celle i SCN indeholder transcriptional aktivator, ur/BMAL1, der fungerer som en heterodimer til inducerer udtryk af PER og græde. PER / CRY kompleks derefter hæmmer funktionen af ur/BMAL1 til at danne en transskription-oversættelse feedback loop, der tager omkring 24 h til fuldstændig2,3.
Tidligere undersøgelser på SCN har hovedsageligt ansat ex vivo SCN skive kultur metode4,5,6 , og mens denne tilgang har givet værdifulde oplysninger, sine begrænsninger har hæmmet vores evne til at få data vedrørende påvirkning af andre hjerne kerner på SCN, samt påvirkning af eksogene stimuli (fxlys) celler bosat i denne vigtige region. I 2001 var Hitoshi Okamura gruppe først til at bruge bioluminescens systemet i vivo genekspression skærm døgnrytmen uret i SCN i frit flytte mus7. Ken-ichi Honma gruppe har tilbragt de sidste par år yderligere at udvikle bioluminescens i vivo optagelse system i SCN8,9,10. Sammen, har disse undersøgelser givet forskerne mulighed for at overvåge døgnrytmen uret i konstant mørke eller efter en lys puls. Men fordi bioluminescens er for svagt til at give mulighed for kontinuerlig overvågning under lys/mørke cyklus, kombineret med det faktum, at lys er den fremherskende signal kræves for SCN-medieret medrivning af døgnrytmen ure11, der er stigende efterspørgsel efter udviklingen af eksperimentelle metoder, der overvinde de begrænsninger i forbindelse med bioluminescens optagelse. Den aktuelle rapport beskriver et fluorescens-baseret system, som blev bygget til at overvåge døgnrytmen uret i SCN- vivo i frit flytte mus. Denne let-at-bruge metode tillader kontinuerlig overvågning under lys/mørke cyklus og giver mulighed for langsigtet observation af transkription af døgnrytmen ur gener i SCN i realtid og med høj tidsmæssige opløsning.

<table border="0" cellpadding="0" cellspacing="0" style="width:1408px;" width="1408">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:627px;">KOD Plus Neo</td>
			<td style="width:403px;">TOYOBO</td>
			<td style="width:133px;">KOD-401</td>
			<td style="width:245px;">Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">pVENUS-N1</td>
			<td>addgene</td>
			<td>#61854</td>
			<td>Plasmid</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">pcDNA3.3_d2eGFP</td>
			<td>addgene</td>
			<td>#26821</td>
			<td>Plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA</td>
			<td>addgene</td>
			<td>#20297</td>
			<td>Plasmid</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MluI</td>
			<td>Thermo Scientific</td>
			<td>FD0564</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EcoRI</td>
			<td>Thermo Scientific</td>
			<td>FD0274</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gibson Assembly Mix</td>
			<td>NEB</td>
			<td>E2611s</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Lipofectamine 2000</td>
			<td>Thermo Scientific</td>
			<td>12566014</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe Filter</td>
			<td>EMD Millipore</td>
			<td>SLHV033RS</td>
			<td>0.45&#160;&#181;m&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HiTrap heparin columns</td>
			<td>gelifesciences</td>
			<td>17-0406-01</td>
			<td>1 mL&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amicon ultra-4 centrifugal filter</td>
			<td>EMD Millipore</td>
			<td>&#160;UFC810024</td>
			<td>100,000 MWCO</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Benzonase nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D5670</td>
			<td>Make fresh solution for each batch</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
			<td>#SR-5M/6M</td>
			<td>Equipment</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">pentobarbital</td>
			<td>SigmaAldrich</td>
			<td>#1507002</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
			<td>#SR-5M/6M</td>
			<td>Equipment</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Hydrogen peroxide solution</td>
			<td>SigmaAldrich</td>
			<td>#216763</td>
			<td>Reagent</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Optical Fiber</td>
			<td>Thorlabs</td>
			<td>FT200EMT</td>
			<td>0.39 NA, &#216;200 &#181;m</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">microsyringe pump</td>
			<td>Nanoliter 2000 Injector, WPI</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ceramic ferrule</td>
			<td>Shanghai Fiblaser</td>
			<td></td>
			<td>230&#8201;&#956;m I.D., 2.5 mm O.D.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gene Observer</td>
			<td>BiolinkOptics</td>
			<td></td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters

Denne teknik kombinerer optisk fiber medieret fluorescens optagelser med præcis levering af rekombinant adeno-associeret virus baseret gen journalister. Denne nye og nemme at bruge i vivo fluorescens overvågning system blev udviklet til at registrere ur gen, Cry1, transcriptional rytme i suprachiasmatic kernen (SCN) af frit flytte mus. At gøre det, en Cry1 transskription fluorescens reporter var designet og pakket ind i Adeno-associeret virus. Renset, koncentreret virus blev sprøjtet ind i musen SCN efterfulgt af indsættelsen af en optisk fiber, som blev derefter fast på overfladen af hjernen. Dyrene var vendt tilbage til deres hjem bure og tilladt en 1-måneds postoperativ nyttiggørelse periode for at sikre tilstrækkelig reporter udtryk. Fluorescens blev derefter optaget i frit flytte mus via en i vivo overvågningssystem, der blev bygget på vores institution. For den i vivo optagelse system, blev en 488 nm laser kombineret med en 1 × 4 beam-splitter, inddelt fire laser excitation udgange lige strøm lyset. Denne opsætning givet os mulighed at optage fra fire dyr samtidig. Hver af signalerne, der udsendes fluorescens blev indsamlet via et photomultiplier tube og et datakort erhvervelse. I modsætning til den tidligere bioluminescens i vivo døgnrytmen ur optagelse teknik tilladt denne fluorescens i vivo optagelse system registrering af døgnrytmen ur genekspression under lys cyklus.

Fluorescens reporter design af Cry1 var show i figur 1A. Ved anvendelse af metode beskrevet ovenfor, 500 nL rAAV-p (Cry1) - intron336 - Venus-NLS-D2 var med held injiceres SCN af en voksen mus og udstillet robust Venus udtryk (figur 1B, 1 C). Fluorescens signaler registreres under 12h / 12h lys/mørke (LD) og mørke og mørke (DD) betingelser (figur 2) givet robuste...

Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Dette nyudviklede fluorescens-baseret teknologi gør det muligt for langsigtet overvågning af transkription af døgnrytmen ur gener i suprachiasmatic kernen (SCN) af frit flytte mus i realtid og med en høj tidsmæssige opløsning.

In Vivo Overvågning af døgnrytmen ur genekspression i mus Suprachiasmatic kernen bruger fluorescens journalister

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Research

JoVE Journal

Neuroscience

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

Ikke-Laser Capture Mikroskopi Tilgang til mikrodissektion af diskrete Mouse Brain Regioner for Total RNA Isolation og downstream næste generation sekventering og Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Høj opløsning Live-Imaging af celle adfærd i udviklingslandene neuroepitel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funktionel Neuroimaging Med Ultrasonic blod-hjerne barrieren forstyrrelser og mangan-forstærket MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Mikromanipuleringer af genekspression i Adult zebrafisk Brain Brug Cerebroventricular mikroinjektion af Morpholino oligonukleotider

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Analyse af genekspression Ændringer i rottehippocampus Efter Deep Brain Stimulation af den forreste thalamiske Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...