Name: large-scale zebrafish embryonic heart dissection for transcriptional analysis
Uploaded: 2015-01-12T13:30:00
Description: Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

Denne protokol følger retningslinjerne i den tyske og Berlin statens lovgivning dyr pleje; zebrafisk håndtering blev overvåget af kommunen for dyrebeskyttelse (LaGeSo, Berlin-Brandenburg). 1. Indhentning af zebrafisk embryoner til Heart Extraction <ol><li> Cross hjerte- reporter zebrafisk såsom Tg (myl7: EGFP) twu34 transgen linie 9 for at opnå embryoner med hjerte-specifikke GFP-ekspression. </li><li> Opretholde embryonerne i ægget vand ved 28,5 ° C, ...

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Denne protokol giver mulighed for hurtig berigelse af zebrafisk embryonale hjertevæv til genekspression analyser. Mængden og kvaliteten af ​​hjerte--specifikke RNA-prøve i høj grad afhænger af et par afgørende trin: først, er mængden af ​​prøven væsentligt forbedret, hvis tab af hjerter forhindres på alle trin i protokollen, da RNA oprensning kun vil arbejde med tilstrækkelig udgangsmateriale. For det andet, renheden af ​​prøven, der afhænger helt af forsøgslederen, bestemmes ved at sortere og...

Zebrafisk (Danio rerio) er meget udbredt i udviklingsmæssige biologi til at studere organogenesis in vivo på grund af sin hurtige, gennemsigtig og ekstrautrin fosterudvikling, kombineret med lille størrelse og tilgængeligheden af transgene reporter linjer med vævsspecifik ekspression af fluorescerende proteiner. Denne lille hvirveldyr er særlig velegnet til at studere hjerte udvikling, fordi iltning af den tidlige zebrafisk embryo ikke er afhængig af hjerterytme og blodgennemstrømning; disse funktioner har tilladt karakterisering af et stort antal cardiovaskulære mutanter 1,2 og zebrafisk er nu almindeligt anerkendt model organisme for at studere hjertesygdomme 3. At studere genekspression under fosterudviklingen, er udskrifter almindeligvis analyseret af hel-mount in situ hybridisering (ønske) 4, RT-qPCR 5, microarrays 6 eller næste generation sekventering (RNA-Seq) 7. MensWISH tillader en rumligt tidsmæssig analyse af genekspression i hele embryo er transkriptniveauer vurderes normalt ved RT-qPCR, microarrays eller RNA-Seq tilgange. Men disse metoder kræver væv berigelse for specifik genekspression profilering. Da den zebrafisk embryonale hjerte udgør en lille brøkdel af hele embryo, transkriptom undersøgelser under hjerte- udvikling kræver en protokol for dissektion og berigelse af hjerter. Desuden, for at opnå fysiologisk relevante data, er det vigtigt at opretholde hjertevævet fuldt funktionel indtil RNA-ekstraktion. Her beskriver vi en protokol for hurtigt at isolere fysiologisk normal og slå hjerter fra hundredvis af zebrafisk embryoner effektivt at opnå høj kvalitet RNA-prøver til yderligere analyser. Den nuværende metode er baseret på den protokol, rapporteret af Burns og MacRae 2006 8. Til berigelse af hjertevæv, begge metoder bruger en transgen myocardial reporter linjeog drage fordel af den differentierede adhæsionsegenskaber af zebrafisk embryonale hjerter versus andre væv. Kort fortalt ved pipettering mange embryoner op og ned gennem en smal pipettespids, hjerter samtidig frigivet fra embryonale organer og efterfølgende adskilt fra embryonale vragrester i to hurtig filtrering trin; fluorescensmærkede hjerter derefter manuelt sorteret fra resterende snavs og opsamles til videre forarbejdning.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr >
			<td >Equipment for raising fish and collecting eggs&#160;</td>
			<td></td>
			<td></td>
			<td>see the Zebrafish Book11 for details</td>
		</tr>
		<tr >
			<td >Fluorescence stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >UV-Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>eg. Thermo Scientific Nanodrop 2000</td>
		</tr>
		<tr >
			<td >Nucleic acid electrophoresis chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dishes 4cm &#216;, coated with 1% agarose in E3 medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipettes and tips (P20, P100, P1000)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1,5ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >15ml and 50ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pair of Dumont #5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >ExactaCruz&#8482; Round Gel Loading Tips in Sterile Rack, 1-200&#956;l&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-201732</td>
			<td></td>
		</tr>
		<tr >
			<td >100 &#956;m filter (BD Falcon 100 mm Cell Strainer)</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr >
			<td >30 &#956;m filter (Pre-Separation Filters-30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >Phase lock gel, heavy, 1,5ml tubes&#160;</td>
			<td>Prime</td>
			<td>2302810</td>
			<td></td>
		</tr>
		<tr >
			<td >Egg water medium</td>
			<td></td>
			<td></td>
			<td>60&#956;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td>
		</tr>
		<tr >
			<td >E3 medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr >
			<td >Tricaine (3-amino benzoic acidethylester)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>4mg/ml Tricaine stock solution, pH 7</td>
		</tr>
		<tr >
			<td >1% agarose (in E3 medium)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1% agarose gel (in TBE buffer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#180;s L-15 medium&#160;</td>
			<td>Gibco</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAlater</td>
			<td>Ambion</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizol</td>
			<td>Ambion</td>
			<td>1559606</td>
			<td></td>
		</tr>
		<tr >
			<td >Glycogen&#160;</td>
			<td>Invitrogen</td>
			<td>10814-010</td>
			<td>20 &#181;g/&#181;L in RNase-free water</td>
		</tr>
		<tr >
			<td >chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >isopropanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >75% ethanol (in DEPC-ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclease-free water or sterilized DEPC treated ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nucleic acid loading buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TBE (Tris/Borate/EDTA) buffer for electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

Zebrafisken embryoniske hjerte er sammensat af kun et par hundrede celler, der kun repræsenterer en lille brøkdel af hele embryoet. Derfor, for at forhindre, at hjerte-transkriptomet bliver maskeret af den globale embryonale transkriptomet, er det nødvendigt at indsamle et tilstrækkeligt antal hjerter for yderligere analyser. Som zebrafisk hjerte-udvikling forløber hurtigt, hjerte indsamling og RNA-ekstraktionsmetoder skal være hurtig for at sikre ensartethed af prøverne. Her præsenterer vi en hurtig manuel dissektion protokol til indsamling funktionelle / slå hjerter fra zebrafisk embryoner. Dette er en væsentlig forudsætning for efterfølgende hjerte--specifik RNA-ekstraktion for at bestemme hjertets specifikke genekspressionsniveauer ved transkriptom analyser, såsom kvantitativ realtids-polymerasekædereaktion (RT-qPCR). Metoden er baseret på differentielle klæbeegenskaber zebrafisk embryoniske hjerte sammenlignet med andre væv; dette giver mulighed for hurtig grafisisk separation af hjerte fra ekstrakardiale væv ved en kombination af strømningsteknisk forskydningskraft afbrydelse trinvis filtrering og manuel samling af transgene fluorescensmærkede hjerter.

Her beskriver vi en repræsentativ hjerte dissektion eksperiment ved hjælp af zebrafisk Tg (myl7: GFP) twu34 transgen linje 9, som udtrykker grønt fluorescerende protein (GFP) udelukkende inden for myocardium (fig. 1). Vi indsamlede både dissekerede hjerter og embryoner, hvorfra de blev afledt til at vurdere renheden af ​​hjertet prøven. Kort fortalt homozygot Tg (myl7: GFP) blev twu34 zebrafisk 9 udkrydsede med vildtype-så alle embryonale h...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

At analysere kardiale genekspressionsprofiler under zebrafisk hjerte udvikling, er den samlede RNA udvindes fra isolerede hjerter. Her præsenterer vi en protokol til opsamling funktionelle / slå hjerter ved hurtig manuel dissektion fra zebrafisk embryoner for at få hjerte-specifikke mRNA.

Storstilet zebrafisk Embryonale Heart Dissektion for Transkriptionel Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

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