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><li>为了准备1X E3胚胎中期，淡化16.5毫升60倍的股票在1升ddiH20。加入100微升1％亚甲蓝。 </li><li>选育<ol><li>宅男和女亲鱼（7 - 18个月）分别以最大限度地提高繁殖力。 </li><li>前2小时，当天鸡蛋所需的前 - 关灯1。建立交配（1男1 - 2名女性）在C罗辛笼如前所述9。渡网箱允许鸡蛋逃脱捕食，因为他们通过在成鱼保持油箱插入的穿孔基地通过。 </li><li>择:对于定时育种，（如，将需要涉及吗啉注射研究），分离雌雄鱼由透明塑料分频器，在上午除去此导致卵受精的定时铺设。对于吗啉的研究，进行功率计算，以评估每次治疗所需的胚胎的鱼的数量。没有效果或变化的幅度的估计，一个初步试验可有n = 8运行。 </li></ol></li><li>倾从交叉笼将鸡蛋打入培养皿（100 - 150胚胎/ 10厘米培养皿）。允许卵沉淀到培养皿的底部，并倒出水。用细尖一次性移液管中取出所有剩余的水。加回步骤1.1取得E3胚胎中期。 </li><li>使自动对焦跛脚抛光一次性吸管为今后的蛋和鱼转移。用剪刀剪毕业一次性移液管以0.25ml毕业。要删除粗糙的边缘，火焰抛光切割端过本生灯。 </li><li>在斑马鱼胚胎的E3介质的培养皿在28.5○C.保持胚胎鱼至4天受精后（dpf）的10厘米每天两次，用火焰打磨毕业一次性移液管，以消除任何未受精的卵子或胚胎死亡（不透明的白色外观）。每天一次，更换E3胚胎中期。倒掉E3胚胎培养基，用细尖一次性移液管除去残留的液体，加回预热（28.5○C）E3胚胎中期。 </li></ol> 2.准备分析解决方案<ol><li>根据使用0.22微米过滤器表1.无菌过滤器测定溶液制备测定溶液。在测定前启动，暖在28.5○C无菌检测解决方案水浴。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td>成分</td><td> ％的总</td><td>卷（微升/ 10毫升） </td></tr><tr><td> 60X E3胚胎中期</td><td> 1.667 </td><td> 166.7 </td></tr><tr><td>双去离子（DDI）H 2 O </td><td> 96.233 </td><td> 9623.3 </td></tr><tr><td>阿尔玛蓝</td><td> 1 </td><td> 100 </td></tr><tr><td> 400毫米氢氧化钠</td><td> 1 </td><td> 100 </td></tr><tr><td> DMSO </td><td> 0.1 </td><td> 10 </td></tr></tbody></table> 表1.分析培养基 3.执行分析<ol><li>冲洗斑马鱼胚胎3次，无菌过滤E3胚胎中期。将所有可行的斑马鱼胚胎从离合器放入烧杯中。无菌过滤器75毫升E3胚胎中期。用细尖一次性移液管，重新从斑马鱼胚胎尽可能将尽可能多的E3胚胎中期的。添加倒退20毫升无菌过滤E3胚胎中期。卸下并更换E3胚胎中期的3倍。 </li><li>板1成鱼每93孔96孔黑色的双面明确的底板。通过单独捕获胚胎内的火焰磨光毕业一次性塑料移液管（步骤1.5），轻轻地喷射到井内的胚胎鱼转移到板上。从转移含有清洗鱼放入3空白孔烧杯中采取E3胚胎中期。这确保了空白孔被暴露了完全相同的水作为含有鱼类的孔中。包含鱼和空白孔两口井应至少半满（200微升）在这一步的结论。 </li><li>更换E3胚胎中期，在所有96个孔，其中包括空白孔检测解决方案。此步骤，应执行1列的时间为每个在96孔板的12个列。使用精尖的一次性反FER吸管，从每一个井板（8孔）的一列去除E3胚胎中期。小心不要碰斑马鱼胚胎。添加300微升测定溶液至每孔从其中除去水。重复使用的板的所有12列测定溶液去除E3胚胎中期和更换。 </li><li>择:为了测试响应于化合物的代谢速度的变化，使一个解决方案，是100倍（100X）所需的治疗浓度。值得注意的是，确保了100倍的溶液不超过10％的DMSO，以限制最终DMSO浓度到不超过0.1％以上。加入3微升100倍的溶液，以各处理井。 注:执行功率计算以评估治疗井的数量。然而，如果没有响应或变化的大小的估计，一个初步试验可在处理8化合物和包含鱼8载体治疗的对照孔中运行。 <ol><li>加入3微升车辆控制来控制（没有DRU摹处理），包含鱼井。为了确保到化合物的荧光响应是的鱼中的动作的结果，应用药物和车辆处理，以3空白孔在96孔板中。 </li></ol></li><li>阅读荧光酶标仪激发波长为530 nm，发射波长为590纳米的鱼填板。 </li><li>将板进入加湿孵化器设置为28.5○C（保持板在黑暗整个试验，以避免测定溶液漂白）。该分析方法可用于持续时间的范围从1小时至24小时运行。测定法的持续时间取决于研究的目标。用于测试响应于短效非稳定化合物短的持续时间可能是最适合的。然而，对于稳定的化合物测试或遗传效应试验，最大限度地提高检测的持续时间最好利用与累积的信号相关的优点。 </li><li>在预先确定的时间点（见步骤3.6，以帮助确定时序）使用相同的设置步骤3.5再读鱼填充板的荧光。 </li></ol> 4.评估在荧光相关的相对变化与治疗<ol><li>井通过从荧光减去荧光在时间0在研究结束时计算变化中的每个的荧光。井无鱼（空格）应包含鱼井还有一些值接近0，远低于价值。 更改荧光=荧光的结论 - 荧光在时间0 </li><li>为了确定在荧光的相对变化，计算在荧光的平均变化对于对照孔，然后通过在对照孔的荧光的平均变化除以在的各孔的荧光变化。值得注意的是，对照可以是载体对照或基因控制。 <img alt="式（1）" src="/files/ftp_upload/53297/53297eq1.jpg" /></li></ol>

<ol>
	<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>

The authors have no competing financial interests to disclose.

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

12.6K 次观看.  Vanderbilt University Medical Center. 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. 

视频: High Throughput Danio Rerio Energy Expenditure Assay

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...