Name: high throughput danio rerio energy expenditure assay
Uploaded: 2016-01-27T17:00:00
Description: Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

注：该协议遵循亚利桑那州办公室的大学研究的负责行为准则和已批准的机构动物护理和使用委员会 1.斑马鱼繁殖和胚胎维护<ol><li>准备E3胚胎中期8。为了使60X贮备液，溶解34.8克氯化钠1.6克氯化钾，氯化钙5.8克2●2H 2 O和9.78克氯化镁 ●6H 2 O在1.95大号ddiH 2 O.调节pH至7.2用NaOH和HCl调节音量用ddiH 2 O 2升</li>...

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	<li>Jao, L. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> PNAS. 110, 13904-13909 (2013).</li><li>Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+knock-in+transgenic+zebrafish+carrying+reporter/driver+genes+by+CRISPR/Cas9-mediated+genome+engineering.">Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.</a> Sci. Rep. 4, 6545 (2014).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Brand, M. D., Couture, P., Else, P. L., Withers, K. W., Hulbert, 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=Evolution+of+energy+metabolism.+Proton+permeability+of+the+inner+membrane+of+liver+mitochondria+is+greater+in+a+mammal+than+in+a+reptile.">Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile.</a> Biochem. J. 275 (Pt 1), 81-86 (1991).</li><li>Renquist, B. J., Zhang, C., Williams, S. Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+assay+for+high-throughput+energy+expenditure+monitoring+in+the+zebrafish.">Development of an assay for high-throughput energy expenditure monitoring in the zebrafish.</a> Zebrafish. 10, 343-352 (2013).</li><li>Livingston, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+volumetric+respirometer+for+long-term+studies+of+small+aquatic+animals.">A volumetric respirometer for long-term studies of small aquatic animals.</a> J.Mar. Biol. Assoc. U.K. 48, 485-497 (1968).</li><li>Makky, K., Duvnjak, P., Pramanik, K., Ramchandran, R., Mayer, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-animal+microplate+assay+for+metabolic+rate+using+zebrafish.">A whole-animal microplate assay for metabolic rate using zebrafish.</a> J. Biomol. Screen. 13, 960-967 (2008).</li><li>Nasiadka, A., Clark, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR J. 53, 161-168 (2012).</li><li>Brand, M., Granato, M., Nusslein-Volhard, C., Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish, A practical approach. , (2002).</li><li>Fry, F. E. J., Hart, 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=Cruising+speed+of+goldfish+in+relation+to+water+temperature.">Cruising speed of goldfish in relation to water temperature.</a> J. Fish. Res. Board Can. 7, 169-175 (1948).</li><li>Oka, T., 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=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC physiol. 10 (21), (2010).</li><li>Song, Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21, 2042-2049 (2007).</li><li>Maddison, L. A., Joest, K. E., Kammeyer, R. M., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+insulin+resistance+in+zebrafish+induces+alterations+in+beta-cell+number+and+glucose+tolerance+in+an+age+and+diet+dependent+manner.">Skeletal muscle insulin resistance in zebrafish induces alterations in beta-cell number and glucose tolerance in an age and diet dependent manner.</a> Am. J. physiol. Endocrinol. , (2015).</li><li>Flynn, E. J., Trent , C. M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+nutritional+control+of+adipogenesis+in+zebrafish+(Danio+rerio).">Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).</a> J. lipid res. 50, 1641-1652 (2009).</li><li>Shimada, Y., Hirano, M., Nishimura, Y., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+fluorescence-based+assay+system+for+appetite-regulating+gene+and+drug+screening.">A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.</a> PloS one. 7, e52549 (2012).</li><li>Tingaud-Sequeira, A., Ouadah, N., Babin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+obesogenic+test:+a+tool+for+screening+molecules+that+target+adiposity.">Zebrafish obesogenic test: a tool for screening molecules that target adiposity.</a> J. lipid res. 52, 1765-1772 (2011).</li></ol>

污染与细菌或真菌将极大地限制了应用此法的。在E3胚胎培养基中的亚甲基蓝限制真菌污染的可能性。在整个胚胎饲养至关重要的是，小心一天两次，每次去除死胚胎。此外，有必要彻底冲洗用无菌的E3胚胎培养基中的胚上测定引发的天。这些2步限制用于胚胎细菌污染的可能性。列入步骤3.2中所述空白孔中可以检测任何细菌污染。细菌污染而产生可测量的信号是罕见的。然而，如果空白孔做显示?...

鼠标是目前肥胖症研究的主要模式。短世代间隔，并在鼠标可用的遗传工具已经无可匹敌的日期。然而，斑马鱼也有一个短的世代间隔（3 - 4个月）和甚至超过了鼠标的易用性基因操纵1,2。斑马鱼保持近90％的哺乳动物的基因，而远远超过了鼠标的后代和潜在的遗传和药物屏幕3的使用数量。 为了开发用于肥胖症的研究斑马鱼模型的潜力，试验必须制定调查影响体重调节，包括能源消耗的因素。作为代谢物通过β氧化和三羧酸循环氧处理被消耗和NADH 2就产生了。因此，NADH 2是代谢物的通过代谢途径（代谢物氧化）的磁通的直接指标。在poikilotherms H +泄漏通过线粒体内膜，该脱开NADH 2氧化从ATP合成，是4 - 5比恒温动物4倍以下。因此，在斑马鱼NADH 2是非常紧密通过氧化磷酸化连接于ATP的产生。在此，我们描述了测量NADH 2生产幼虫斑马鱼作为能量消耗5代理的检测。 耗氧量的黄金标准测量的能量消耗。然而，为了最好的利用斑马鱼的高通量的潜力，能量消耗的测定必须是适合于高通量。依赖于一个封闭的腔室循环系统氧消耗系统的吞吐量由可用6室的数目的限制。露天氧气消耗量/ CO 2的生产试验也已在斑马鱼5,7应用。这些开放空气96孔板基D系统适于高吞吐量。不幸的是，与环境进行气体交换限制这些测定的灵敏度。我们最近公布了使用氧化还原指示剂的AlamarBlue 5监视NADH 2的检测中的应用。该测定中克服了吞吐量和共同到氧消耗在斑马鱼的分析灵敏度的限制。 斑马鱼正在成为整个身体能量平衡的研究日益重要的模式。在某种程度上，因为斑马鱼是适合于向前遗传筛选和药物屏幕使用。此外，有针对性的遗传操作，包括敲除和敲入，可以快速应用。之前我们已经表明，这种检测可以与浴药的应用和基因敲除或基因敲除结合起来，以确定化合物的基因，改变代谢率5。此外，该试验被设计用于利用固有的斑马鱼的高通量的优点。 </P&gt;

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

斑马鱼是一种重要的模式生物与必须使斑马鱼的能量平衡和肥胖的研究中广泛应用模式的潜在先天的优势。斑马鱼的小尺寸允许在胚胎测定以在96或384孔板形式进行，基于吗啉代和CRISPR技术促进易于遗传操作，和药物治疗通过浴的应用是可行的。此外，斑马鱼是理想的正向遗传学屏幕允许新基因的发现。定斑马鱼的相对新颖作为模型肥胖，有必要开发工具，充分利用这些好处。在此，我们描述了一个同时测量数千胚胎斑马鱼的能量消耗的方法。我们已经开发出一种整体动物微孔板平台，其中，我们使用96孔板来分离单个鱼和我们评估使用市售的细胞培养物生存力重新累积NADH 2生产代理的AlamarBlue。在变温动物NADH 2产生和能量消耗之间的关系是紧密相连。这种能量消耗测定创建快速筛选药理学或遗传操作直接改变能量消耗或改变响应于施加的药物（例如，胰岛素增敏剂）的潜力。

测定溶液不会在没有胚胎鱼（图1）增加的荧光。然而，该测定是在井内代谢率的微小变化高度敏感。内一个井的单个鱼产生的信号在1小时内（P &lt;0.0001）显著不同于空白孔中。 图2提供了孵育24小时后通过胚胎鱼产生的信号的视觉表示。对于这张照片的目的明确的双面井使用。然而，黑色双面井优选用于测定性能，以防止来自附近孔的荧光信号的浸出。 <p class="jove_cont...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

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High Throughput Danio Rerio Energy Expenditure Assay

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