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.

该协议遵循了德国和柏林国家法律的动物护理准则;斑马鱼的处理是由动物保护（LaGeSo，柏林 - 勃兰登堡）地方当局监控。 1.获取斑马鱼胚胎的心脏提取<ol><li>横心脏记者斑马鱼，例如作为Tg（MYL7：EGFP）转基因twu34线9，以便获得胚胎心脏特异性GFP表达。 </li><li>保持水蛋胚胎在28.5°C，直到所需的萌芽阶段10,11。确保所收集的胚胎人口是同?...

<ol>
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J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Brownlie, A., 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=Characterization+of+embryonic+globin+genes+of+the+zebrafish.">Characterization of embryonic globin genes of the zebrafish.</a> Dev Biol. 255, 48-61 (2003).</li><li>Marvin, 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=Developmental+expression+patterns+of+the+zebrafish+small+heat+shock+proteins.">Developmental expression patterns of the zebrafish small heat shock proteins.</a> Dev Dyn. 237, 454-463 (2008).</li><li>Lam, C. S., Marz, M., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gfap+and+nestin+reporter+lines+reveal+characteristics+of+neural+progenitors+in+the+adult+zebrafish+brain.">gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain.</a> Dev Dyn. 238, 475-486 (2009).</li><li>Wang, 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=Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis.">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li><li>Heiman, 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=A+translational+profiling+approach+for+the+molecular+characterization+of+CNS+cell+types.">A translational profiling approach for the molecular characterization of CNS cell types.</a> Cell. 135, 738-748 (2008).</li><li>Tryon, R. 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该协议允许斑马鱼胚胎心脏组织的快速富集的基因表达分析。心脏特异性RNA样品的数量和质量在很大程度上取决于几个关键步骤：第一，如果能够防止在该协议中的每一步的心损耗的样品的量有很大的提高，因为RNA纯化将仅具有足够的工作起始材料。第二，样品，这完全取决于实验者的纯度，通过分类和收集的心内的培养皿同时严格排除其它组织确定的。第三，样品的质量主要取决于限制可能发?...

斑马鱼（ 斑马鱼 ）被广泛用于发育生物学研究器官体内 ，由于其快速的，透明的和宫外胚胎发育，结合小尺寸和转基因记者线荧光蛋白的组织特异性表达的可用性。这个小脊椎动物特别适于研究心脏发育，因为早期的斑马鱼胚胎的氧合不依赖于心脏的跳动及血液流动;这些特点使大量心血管突变1,2的表征和斑马鱼现在是一个被广泛认可的模式生物来研究心脏疾病3。 到胚胎发育过程中研究基因表达，转录通常通过整个安装原位杂交（WISH）4分析，RT-qPCR的5，微阵列6，或下一代测序（RNA测序）7。而WISH允许基因表达的整个胚胎内的空间 - 时间分析，转录水平通过RT-qPCR的，微阵列或RNA测序方法，通常进行评估。然而，这些方法需要组织富集为特定的基因表达图谱。 因为斑马鱼胚胎心脏表示整个胚胎的一小部分，心脏发育过程中转录研究需要一个协议，用于解剖和心脏的富集。此外，为了得到生理上相关的数据，以维持心脏组织完全功能，直到RNA提取它是很重要的。在这里，我们描述了一种协议，用于快速分离生理正常和跳动心脏从数百斑马鱼胚胎的有效地获得高品质的RNA样品用于进一步分析。本方法是基于报道的烧伤和麦克雷，2006年8所述的协议，对于心脏组织的富集，这两种方法都使用的转基因心肌记者线并利用斑马鱼胚胎心脏与其他组织的鉴别粘附性能优势。简要地说，通过一个狭窄的枪头吹打许多胚胎上下，心脏被同时从胚体释放并随后从胚胎碎片两个快速过滤步骤中分离;荧光标记的心脏，然后手动地从剩余的碎片来分类并收集用于进一步处理。

<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

斑马鱼胚胎的心脏是由只有几百细胞，占整个胚胎中只有一小部分。因此，为了防止被掩蔽由全局胚胎转录心脏转录，有必要收集足够数量的心作进一步的分析。此外，如斑马鱼心脏发育迅速进行，心脏收集和RNA提取方法需要快，以保证样品的均匀性。在这里，我们提出了一个快速的手动清扫协议收集功能/跳动的心，从斑马鱼胚胎。这是为随后的心脏特异性RNA提取，以确定心肌特异性基因表达水平通过转录组分析，例如定量实时聚合酶链反应（RT-qPCR的）的必要先决条件。该方法是基于在斑马鱼胚胎心脏和其他组织相比的差的粘合性能;这允许迅速的phy从心外心脏组织由流体剪切力的破坏，逐步过滤和人工采集转基因荧光标记心中的组合SICAL分离。

在这里，我们描述了使用斑马鱼的Tg（MYL7：GFP）的有代表性的心脏解剖实验专在心肌（图1）的转基因twu34线9，它表达绿色荧光蛋白（GFP）。我们收集了两个解剖心，从他们得出评估心脏样品的纯度胚胎。简言之，将纯合的Tg（MYL7：GFP）twu34斑马鱼9进行异型杂交用野生型，使得所有的胚胎心脏的GFP标记。大约500胚胎（56 HPF）转移到1.5毫升管（约1...

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

到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。

大规模的斑马鱼胚胎的心脏解剖的转录分析

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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Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

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Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

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Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

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幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

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The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
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Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

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