Name: use of the teton system to study molecular mechanisms of zebrafish regeneration
Uploaded: 2015-06-25T13:00:00
Description: Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Forfatterne takker Christa Haase, Doris Weber og Brigitte Korte for teknisk bistand. Arbejdet i Weidinger lab er støttet af tilskud fra Deutsche Forschungsgemeinschaft WE 4223 / 3-1, WE 4223 / 4-1 og af Deutsche Gesellschaft für Kardiologie via en Oskar-Lapp-Stipendium og Klaus-Georg-und-Sigrid- Hengstberger-Forschungsstipendium.

1. generation af transgene TetActivator Fish Lines <ol><li> Generering af TetActivator konstrukt <ol><li> Clone regulatoriske sekvenser af interesse opstrøms for TetActivator kassetten i vektor # 1247 anvendelse af standardteknikker. Alternativt bruge rekombinationsteknikker til at generere en BAC, hvor TetActivator kassetten (vektor # 1180) indsættes i det første exon af målgenet, og hvor Tol2 inverterede gentagelser indføres i BAC rygrad (for en detaljeret recombineering protokol se 7,8). </li><li> F...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+kit+ligand+a+functional+expression+reveals+lineage-specific+competence+to+promote+melanocyte+rescue+in+the+mutant+regenerating+caudal+fin.">Clonal analysis of kit ligand a functional expression reveals lineage-specific competence to promote melanocyte rescue in the mutant regenerating caudal fin.</a> PloS one. 9 (7), e102317 (2014).</li><li>Wehner, 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=Wnt/beta-catenin+signaling+defines+organizing+centers+that+orchestrate+growth+and+differentiation+of+the+regenerating+zebrafish+caudal+fin.">Wnt/beta-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.</a> Cell Rep. 6 (3), 467-481 (2014).</li><li>Huang, C. J., 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=Conditional+expression+of+a+myocardium-specific+transgene+in+zebrafish+transgenic+lines.">Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.</a> Dev Dyn. 233 (4), 1294-1303 (2005).</li><li>Knopf, F., 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=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li><li>Provost, E., Rhee, J., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li><li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish.">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Kim, J. 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6 (4), e18556 (2011).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 53 (2), 192-204 (2012).</li><li>Hyde, D. R., Godwin, A. R., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, V., 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=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. 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Den voksne zebrafisk har en forbløffende evne til med succes at regenerere mange indre organer og vedhæng. En grundig forståelse af de molekylære og cellulære mekanismer involveret kræver vævsspecifik analyse af gen funktioner og signalveje. Mod dette, Teton system giver et effektivt redskab til spatiotemporally kontrolleret genekspression i embryonale og voksne zebrafisk. Teton systemet konstruktioner og metoder, der er beskrevet i dette manuskript har været anvendt med succes i en nylig undersøgelse af vores ...

Zebrafisken er en veletableret hvirveldyr model organisme for at studere mange aspekter af udvikling, fysiologi, sygdom og regenerering. Med den stigende vedtagelsen af ​​zebrafisk som en model for post-embryonale biologiske processer, eksperimentelle værktøjer til inducerbar, vævsspecifik manipulation af genekspression eller signalveje er blevet stadig vigtigere. Især har undersøgelser i orgel og vedhæng regenerering hos voksne zebrafisk lidt under manglende af værktøjer til dissektion af spatio-temporale krav signalveje i disse regenerative processer. I øjeblikket har tre forskellige systemer blevet anvendt til at opnå betinget, vævsspecifik genekspression i regenererende organer voksne zebrafisk: Cre-lox-systemet, mosaik ekspression af varmechok-inducerbare transgener ved hjælp transposon-medieret somatisk transgenese, og Teton systemet 1 -3. Teton refererer til en variant af et tetracyclin-contrullet transkriptionel aktiveringssystem, hvor ekspression er aktiveret i nærvær af antibiotikummet tetracyclin eller et derivat, f.eks doxycyclin. Cre-lox-systemet, som det hidtil har været anvendt i voksne fisk, bygger på en Tamoxifen-kontrollerede Cre rekombinase (CreERT2), hvis ekspression er rumligt begrænset af vævsspecifikke regulatoriske elementer. Cre-drevet fjernelse af et STOP kassette letter ekspression af genet af interesse drevet af en promotor, der skal være aktiv i alle celletyper 1. Transposon-medieret oprettelse af mosaically udtrykte somatiske transgener tilvejebringer et system til inducerbar transgenekspression i individuelle cellelinier. Injektion af zebrafisk embryoner med et Tol2 transposon bærer et gen af interesse under transkriptionel kontrol af en varmechokpromotor resulterer i kimære individer typisk bærer transgenet kun i diskrete cellelinier af en regenererende organ 2. Mens begge systemer giver mulighed for betinget væv-specifikke genekspression, Cre-lox-systemet er ikke reversibel, og strategien ved anvendelse transposonbaseret klonal mærkning lider sin stokastiske natur. Således har vi og andre for nylig tilpasset transgene Teton systemer til brug i zebrafisk, som letter tidsmæssigt og rumligt kontrolleret genekspression, der er desuden justerbar og reversibel 3-5. Teton systemet her anvendt omfatter en transgen driver linie (TetActivator), hvori en Doxycyclin (DOX) -inducerbare transkriptionel aktivator (forbedret omvendt tetracyclin transaktivator, irtTA, kort- TETA) er under kontrol af vævsspecifikke genomiske regulatoriske sekvenser. For det andet kræver en transgen responder linje (TetResponder), som huser et gen af interesse under transkriptionel kontrol af tetracyclin operatør (Tet responselement; Tetre) (figur 1A). Anvendelsen af ​​specifikke kombinationer af TetActivator og TetResponder linjer giver mulighed for betinget væv-specific manipulation af genekspression. Vi har for nylig brugt Teton system til at sondere for vævsspecifikke funktioner Wnt / beta-catenin signalering i den voksne regenererende zebrafisk halefinnen 3. I protokollen beskrevet her beskriver vi en arbejdsgang til opsætning og brug af Teton-system i zebrafisk, især for studier af fin regenerering. Dette omfatter detaljerede instruktioner om, hvordan at generere stabile transgene TetActivator og TetResponder linjer og en protokol for transgen induktion i embryoner og voksne zebrafisk. Desuden beskriver vi teknikker til kontrol af vævsspecifik genekspression i fin regenerere, herunder en protokol til fremstilling af kryosnit af voksne zebrafisk finner. Derudover diskuterer vi overvejelser for udformningen af ​​TetActivator transgen, valget af en transgenese metode, og detektion af TetResponder ekspression. Derfor er det overordnede mål med denne protokol er at tjene som et blueprinttil oprettelse et funktionelt teton system zebrafisk at opnå betinget vævsspecifik genekspression, som kan anvendes på enhver væv af interesse. Vi har skabt en TetActivator vektor tillader I-Scel eller Tol2-medieret dannelse af stabile TetActivator linjer ved hjælp af korte genomiske regulatoriske sekvenser (forstærker fragmenter; Weidinger lab plasmid database nej 1247;. Figur 1B). Denne konstruktion indeholder en TetActivator kassette bestående af M2 mutant variant af den omvendte Tet-repressor domæne fusioneret med Herpes simplex virus VP16-transaktiveringsdomænet-derivat 3F [irtTAM2 (3F)]. Ekspression af TetActivator (TETA) kan let overvåges, da det er co-udtrykkes med fluoroforen AmCyan fra samme åbne læseramme; en P2A peptid medierer ribosomale springe, hvilket skulle resultere i produktion af TETA og AmCyan som særskilte proteiner ved en 1: 1-forhold 5,6. Konstruktionen indeholder også en poylinker 5 &#39;af TetActivator kassette at lette indføring af genomiske regulatoriske sekvenser af interesse under anvendelse af konventionelle kloningsmetoder. Derudover har vi skabt en konstruktion, der består af den ovenfor beskrevne TetActivator kassette plus en kanamycinselektion kassette (Weidinger lab plasmid database nr 1180;. Figur 1C), som kan rekombineres ind i en bakterielt kunstigt kromosom (BAC), der indeholder en stor genomisk region ( sædvanligvis i startkodonen af ​​et gen, hvis ekspressionsmønster skal efterlignes af transgenet). Begge konstruktioner er tilgængelige fra Weidinger laboratoriet efter anmodning.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breeding boxes</td>
			<td>Aqua Schwarz</td>
			<td>AquaBox 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>e.g. Leica, Thermo-Scientific</td>
			<td>varies with the manufacturer</td>
			<td>for cryosectioning</td>
		</tr>
		<tr>
			<td>4&#8217;, 6- diamidino-2-phenylinodole (Dapi)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use 1/5000 in PBS 
			for visualization of nuclei</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM) 
			for TetResponder induction</td>
		</tr>
		<tr>
			<td>E3 embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2&#903; 2 H2O, 0.33 mM MgSO4&#903;7 H2O, 0.2 &#8240; (w/v) methylene blue, pH 6.5 
			for embryo/larvae husbandry</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>4 % (w/v) paraformaldehyde in PBS, pH 7.5 
			for fixation</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline (PBS)</td>
			<td></td>
			<td></td>
			<td>1.7 mM KH2PO4, 5.2 mM Na2HPO4, 150 mM NaCl, pH 7.5</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline + Tween 20 (PBT)</td>
			<td></td>
			<td></td>
			<td>1x PBS with 0.1 % Tween 20</td>
		</tr>
		<tr>
			<td>Superfrost Ultra Plus adhesion microscope slides</td>
			<td>Thermo Scientific&#160;</td>
			<td>1014356190</td>
			<td>for collection of tissue sections</td>
		</tr>
		<tr>
			<td>Stereo fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>for fluorescence-based genotyping</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>e.g. Biorad, Applied Biosystems</td>
			<td>varies with the manufacturer</td>
			<td>for PCR-based genotyping</td>
		</tr>
		<tr>
			<td>Tissue freezing medium (TFM)</td>
			<td>Triangel Biomedical Sciences</td>
			<td>TFM-C</td>
			<td>for embedding of tissue samples</td>
		</tr>
		<tr>
			<td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>for anesthesia 
			use at 1 mg/ml in E3 embryo medium</td>
		</tr>
	</tbody>
</table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

At etablere et funktionelt teton system til vævsspecifik inducerbar genekspression, transgene TetActivator og TetResponder linjer skal genereres (figur 1A). Dette opnås ved mikroinjektion TetActivator (figur 1B - C) eller TetResponder (figur 1E) konstruktioner i tidlige zebrafisk embryoner og efterfølgende integration kim-linie. Funktionelle TetActivator konstruktioner kan enten genereres ved kloning af korte regulatoriske sekvenser (enhancerelemente...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Anvendelse af Teton System til Undersøgelse molekylære mekanismer for zebrafisk Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

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