Name: an optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions
Uploaded: 2018-03-22T14:00:00
Description: Watch this Scientific Journal Video about An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions at JoVE.com

Dette arbejde blev støttet af JST, PRESTO (AI), Core forskning for Evolutional videnskab og teknologi (JPMJCR12W2 (Lisbeth)), licensbetaling for videnskabelig forskning på Innovative områder (Undervisningsministeriet, kultur, sport, videnskab og teknologi (MEXT), Japan 26119708 (AI) og 16 H 06480 (Lisbeth)), videnskabelig forskning (et) (Japan samfund til fremme af videnskab (JSP'ER) 24240049 (Lisbeth)), og unge forskere (A) (JSP'ER 15 H 05326 (AI)), og en licensbetaling for videnskabelig forskning på Innovative områder "fluorescens Live imaging"MEXT, Japan og Platform for dynamiske tilgange til levende System fra MEXT, Japan.

1. generation af stabil cellelinjer af Tol2 systemet
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Transfect plasmid vektorer (figur 1A) af Tol2-baseret optogenetic moduler sammen med transposase (Tol2) udtryk vektor (pCAGGS-mT2TP) i C2C12 celler. I alle trin, kultur celler med DMEM medium suppleret med 10% føtal bovint serum (FBS) og penicillin-streptomycin ved 37 ° C (tabel 1), i nærværelse af 5% CO2, ellers betegnet.
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<li>Tæl trypsinized celler med en celle-counter og plade 5 x 10...

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Vi viste en metode til at styre gen expression dynamics med en hyppighed på 2 til 3 h. Denne tidshorisont er meget kortere end dem i andre konventionelle systemer, herunder Tet-On-system og den oprindelige Niels. Vigtige parametre til at nå de ultradian tidsskalaer er halveringstider foto-induceret molekylære produkter, mRNAs og proteiner. Disse kinetiske parametre kan afhænge af celletyper og arter. For tuning forsvindingskinetik, er erstatter Hes1 3' UTR sekvenser med andre en straight-forward måde, fordi det ikke...

Celle-til-celle kommunikation spiller en kritisk rolle i embryonale mønstre i udviklingsprocesser. Hvirveldyr embryoner udgøres de metameriske strukturer, der kaldes somites langs anterior-posterior krop akse med en præcis tidsmæssige nøjagtighed under kontrol af en tid-holder ur, kaldet segmentering uret1. Under denne proces, en gruppe af presomitic mesoderm (PSM) celler regelmæssigt omdannes til somites i en synkron måde. Denne proces indebærer synkroniserede oscillerende genekspression og PSM celler, der svinger i fase udgør de samme somites. Perioden i den oscillerende genekspression er omkring 2 til 3 timer i mus og ca 30 min i zebrafisk. Når adskilles, PSM celler mister synchrony2,3, men når de er re aggregerede, de kan selv organisere og gendanne den befolkning synchrony4, tyder på, at celle-celle kobling er en nøgle til den synkroniserede svingninger.
Omfattende indsats afslørede, at signaling molekyler i Delta-Notch pathway er tæt forbundne til synkroniserede svingninger af segmentering ur gener. Enten farmakologiske hæmmere eller genetiske mutationer af Notch signalering desynchronize befolkningen i oscillatorer. Mutanter af Notch signalering komponenter, såsom DeltaC, DeltaD og Notch1a, vist i zebrafisk, asynkron svingninger5,6. Med kylling eller mus embryoner er ikke kun Notch ligand Delta-lignende1 (Dll1), men også Notch Modulator Lunatic fringe (Lfng) påkrævet for synkroniseret svingninger7,8,9. Men det har været svært at teste den funktionelle kapacitet af sådanne molekyler til dynamisk informationsoverførsel fra celle til celle, fordi tidsmæssige resolutioner af konventionelle undertrykkelse af netbårne gen forordning dynamik ikke var tilstrækkelige til at undersøge de processer af tidsskalaer af 2-3 h (ultradian rytmer).
Vi har for nylig udviklet en integreret metode til at kontrollere og overvåge gen expression mønstre i pattedyrceller10. Denne teknologi gør det muligt for induktion af gen expression pulser af periodiske lys belysning på ultradian tid-skalaer. Denne protokol repræsenterer metoder at etablere lysfølsomme cellelinjer og observere dynamisk svar af reporter celler af live-celle luminescence overvågning i sammenhænge af celle-til-celle kommunikation. Denne metode gælder for analysen af mange andre signaling veje.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">FACS</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:180px;">FACSAriaII SORP</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Camera</td>
			<td>Andor</td>
			<td>iKon M-934</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>IX-81 ZDC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PMT device</td>
			<td>Churitsu eletric corp.</td>
			<td>CL24B-LIC/B</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Blue LED illuminator</td>
			<td>OptoCode</td>
			<td>LEDB-SBOXH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td>Nacalai</td>
			<td>08459-35&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin-streptomycin</td>
			<td>Nacalai</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal bovine serum</td>
			<td>Sigma</td>
			<td>172012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KRYSTAL24 (black 24 well plate )</td>
			<td>Hi-tech</td>
			<td>303012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">D-Luciferin Potassium Salt</td>
			<td>Nacalai</td>
			<td>20028-24&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Light meter</td>
			<td>LI-COR Biosciences</td>
			<td>LI-250A</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">anti-HA-Peroxidase antibody</td>
			<td>Roche</td>
			<td>clone 3F10</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">anti-Actin-Peroxidase antibody</td>
			<td>Wako</td>
			<td>clone 2F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions

Celler bør reagere korrekt på tidsligt skiftende miljøer, som påvirkes af forskellige faktorer fra de omkringliggende celler. Notch signalering pathway er en af sådanne væsentlige molekylære maskineri til celle-til-celle kommunikation, som spiller vigtige roller i den normale udvikling af embryoner. Denne vej indebærer en celle til celle overførsel af oscillerende oplysninger med ultradian rytmer, men trods fremskridt i molekylærbiologiske teknikker, har det udfordrende for at belyse virkningerne af flercellede interaktioner på oscillerende gen Dynamics. Vi præsenterer her, en protokol, der tillader optogenetic kontrol og live overvågning af gen expression mønstre tidsmæssige præcist. Denne metode afslørede med held at intracellulære og intercellulære periodiske indgange af Notch signalering entrain iboende svingninger af frekvens tuning og fase skiftende den encellede opløsning. Denne fremgangsmåde gælder for analysen af de dynamiske funktioner i forskellige signaling veje, giver en unik platform for at teste en funktionel betydning af dynamiske gen expression programmer i flercellede systemer.

Vi tilpasset Niels system11,12, som giver mulighed for foto-induceret genekspression i pattedyrceller til studiet af genetiske oscillatorer med 2 - 3 h hyppighed. Dette system består af to dele: foto-inducerbar transcriptional activator hGAVPO og en UAS-promotor kassette til drev transskription af vilkårlige gener af interesse. For at fremskynde den pulsatile kinetik af foto-induceret genekspression, blev polyA sekvens i UAS-pro...

Watch this Scientific Journal Video about An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions at JoVE.com

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Vi præsenterer her, en protokol for at analysere celle til celle overførsel af oscillerende oplysninger optogenetic kontrol og live overvågning af genekspression. Denne fremgangsmåde giver en enestående platform for at teste en funktionel betydning af dynamiske gen expression programmer i flercellede systemer.

En Optogenetic metode til at styre og analysere gen Expression mønstre i celle-til-celle interaktioner

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

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