作者要感谢迪肯大学斑马鱼设施的工作人员提供了良好的持续牧护理。将YG支持阿尔弗雷德·迪肯博士后研究奖学金和中央迪肯大学（Deakin University）的研究资助。 SLM NHMRC职业发展奖学金的支持。 ACW是支持的的NHMRC启用补助金。所有的作家都迪肯大学的分子医学研究所战略研究中心的支持。

第1部分:化学处理的斑马鱼胚胎<ol><li>收集斑马鱼胚胎在受精后。孵育在28.5℃下在E3介质的100×15毫米的培养皿中。 注:如果在 原位杂交或油红O染色，1 -苯基-2 -硫脲（PTU）的最终浓度为0.2 mM的被添加到抑制色素形成1，但不是必要的海马分析。 </li><li>药理研究，在适当的斑马鱼胚胎在受精后26小时左右（HPF）的终浓度为所需的化学添加一个合适的工具，唯一的控制。 注意:光敏感的药物抑制剂，胚胎可以被允许发展在分析前的暗。 </li></ol>第2部分:现场代谢个人使用海马XF 24分析仪<ol><li>每次运行前，海马XF 24分析仪墨盒，里面的O 2和H <s高达&gt; +荧光基团是通过一个自动执行的处理的分析仪校准。 </li><li>制备24孔XF 24胰岛板。采用下面的实验序列: <ol><li>由于氧的消耗是敏感的温度波动，四个井，用于控制整个板的可能的温度波动。这些井的不包含胚胎，但都充满了700微升E3类介质中（ 图2A）。 </li><li>余下的20井（见2.2部分）填充E3类介质和每一个胚胎（ 图2B）与700微升。 </li><li>对于每个实验，10的胚胎与车辆只（对照组）和10用化学抑制剂（处理的）处理。交替控制和处理的胚胎（ 例如 A2:控制胚胎;以及A3:处理过的胚胎以及A4:控制胚胎，治疗以及A5胚胎等）。 </li><li>一个小岛拍摄屏幕的顶部添加每个孔中，以确保该标本保持在整个试验中（ 图2B）的测量室。 注:化学处理的胚胎保持其原始的化学溶液在整个过程中，没有需要洗出前的化学运行Seahorse的分析仪程序。 </li><li>一旦完成后，板装入Seahorse的分析仪，并开始测定。 </li></ol></li><li>样本分析。两种不同的检测常规进行。 <ol><li>的基础呼吸/最大呼吸计划（持续时间约90分钟）。基础呼吸的改变支撑，而最大的呼吸代谢功能障碍，是衡量能源生产总量的能力，在此参数与一些病理和生理状态的改变。的备用呼吸能力，或系统，以进一步提高ATP生产的能力，同时也计算出从本次测试的减法的基础，从最大的呼吸。三重复的每个组合（2分钟）/（1分钟）/测量等待周期（2分钟），先建立基础的呼吸。在第三个周期的结束，使最终浓度为2.5μM的FCCP（线粒体protonophore /解偶联剂）被添加到每个孔中允许测量的最大呼吸。然后，在测量周期重复5〜8倍（ 图3）。 </li><li> ATP营业额/的质子漏程序（持续时间约60分钟）。呼吸，由于ATP营业额的主要功能，线粒体的ATP生产的形式，而由于质子漏，或脱钩呼吸呼吸，与其他参数的线粒体的功能，包括基础呼吸和活性氧物种的形成有着千丝万缕的联系。呼吸由于ATP周转后，加入25μM的寡霉素（抑制剂的A表示的呼吸中的差异TP酶）相比，基础呼吸的影响。非耦合呼吸，或呼吸由于质子泄漏，确定通过计算25μM鱼藤酮（一个复杂的I抑制剂）的添加后，寡霉素-介导的呼吸和呼吸之间的差异。每个线粒体抑制剂加入后，重复测量周期的8倍。 </li></ol></li></ol>的运行结束时，计算平均值10控制和10个化学处理的胚胎。 第3部分:油红O染色<ol><li> 50斑马鱼胚胎在dechorionated用细镊子和4％PFA固定于4℃过夜</li><li>进行油红O染色，如前面所述2（参见图4）。 </li></ol>

<ol>
	<li>N&#252;sslein-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. , (2002).</li><li>Schlombs, K., Wagner, T., Scheel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-1+protease+is+required+for+cartilage+development+in+zebrafish.">Site-1 protease is required for cartilage development in zebrafish.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 14024-14029 (2003).</li><li>Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+power+of+the+zebrafish+for+disease+analysis.">The power of the zebrafish for disease analysis.</a> Hum. Mol. Genet. 18, R107-R112 (2009).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Stackley, K. D., Beeson, C. C., Rahn, J. J., Chan, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profiling+of+zebrafish+embryonic+development.">Bioenergetic profiling of zebrafish embryonic development.</a> Plos One. 6, e25652 (2011).</li><li>McGee, S. L., Sadli, N., Morrison, S., Swinton, C., Suphioglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DHA+protects+against+zinc+mediated+alterations+in+neuronal+cellular+bioenergetics.">DHA protects against zinc mediated alterations in neuronal cellular bioenergetics.</a> Cell Physiol. Biochem. 28, 157-162 (2011).</li><li>Sivitz, W. I., Yorek, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+diabetes:+from+molecular+mechanisms+to+functional+significance+and+therapeutic+opportunities.">Mitochondrial dysfunction in diabetes: from molecular mechanisms to functional significance and therapeutic opportunities.</a> Antioxid. Redox Signal. 12, 537-577 (2010).</li><li>Bournat, J. C., Brown, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+obesity.">Mitochondrial dysfunction in obesity.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 446-452 (2010).</li><li>Ghafourifar, P., 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=Mitochondria+in+multiple+sclerosis.">Mitochondria in multiple sclerosis.</a> Front Biosci. 13, 3116-3126 (2008).</li><li>Winklhofer, K. F., Haass, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+Parkinson's+disease.">Mitochondrial dysfunction in Parkinson's disease.</a> Biochim. Biophys. Acta. 1802, 29-44 (2010).</li><li>Maruszak, A., Zekanowski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+Alzheimer's+disease.">Mitochondrial dysfunction and Alzheimer's disease.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 35, 320-330 (2011).</li><li>de Moura, M. B., dos Santos, L. S., S, L., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+neurodegenerative+diseases+and+cancer.">Mitochondrial dysfunction in neurodegenerative diseases and cancer.</a> Environ. Mol. Mutagen. 51, 391-405 (2010).</li></ol>

没有利益冲突的声明。

斑马鱼是一种行之有效的遗传模型的正向（ENU诱变）和反向（耕，锌指核酸酶，有针对性的基因敲除，吗啉击倒）遗传方法3,4，而在斑马鱼胚胎的基因的功能也可以很容易地阻止或激活使用特定的编码产物的选择性药效成分。由于它们的外部的发展和小尺寸，斑马鱼胚胎是特别适合于代谢分析。然而，在斑马鱼胚胎在体内的代谢和线粒体功能强大的测量，只有一个初步的说明报告5。海马分析最初被设计用于基于细胞的代谢研究，并已被证明，得到准确和可靠的结果6。这种新方法的应用斑马鱼的胚胎是重要的，并可能增加这种模式的代谢研究的更广泛的使用。在这项研究中，我们展示了在斑马鱼胚胎海马分析仪，包括基础呼吸，最大呼吸，备用呼吸能力，ATP营业额和质子漏的呼吸参数的测量。我们还提供这样的测量可以如何与其他生理参数，在这种情况下，脂质蓄积，并在该测定系统中使用的药理抑制剂相关的一个例子。结合使用基因改变胚胎，这提供了一个有力的实验平台，为了解对新陈代谢的影响因素。 有很多种为这个新的方法的应用程序，与线粒体功能障碍被牵连在许多人类的疾病，如糖 ​​尿病7 8，肥胖，多发性硬化症9，帕金森氏病10，阿尔茨海默氏病11和某些类型的癌症12。重要的是，O我们的工作是在体内 ，在那里所有的环境的影响-如细胞因子，与发展有关的生长等-是活跃的，从而提供了一种在体内的呼吸和代谢更新有关生理视图。由于化学屏幕也经常在野生型和突变的背景斑马鱼胚胎（ 图1），小说，影响呼吸，线粒体功能或代谢海马分析仪，可以很容易地确定使用的药品。使用Seahorse的分析仪产生的结果可与其他实验中使用，以提供额外的信息。这可以包括生理的分析，如油红染色，或分子生物学分析，如原位杂交特定的脂肪细胞的的标记如cebpα，PPARα，PPARγ，FAS等。 这种方法仍然有一定的局限性。虽然我们和其他人能够使用寡测量余吕杏茜的ATP生产和质子漏幼胚（ 见图3），在胚胎年龄超过60 HPF寡治疗是无效的。我们相信，在旧的胚胎寡不能扩散，容易在年轻的胚胎，结果无法解释。由于寡结果的测定呼吸由于ATP的营业额和非耦合呼吸，是强制性的，我们目前正在研究高浓度的寡旧胚胎。然而，抗霉素A治疗仍然有效的更长的时间，与其它的测量，如基础呼吸，最大呼吸和备用呼吸能力，可以在旧的斑马鱼胚胎，最多至68 hpf时（未示出）。 在使用当前的海马分析仪设置的另一个限制是每个胰岛捕捉板的物理空间内，使得它们只适合斑马鱼胚胎和年轻幼虫。因此，执行metabolIC饮食引起的肥胖的成年鱼的研究，例如，尚未技术上是可行的。不过，这项研究可能会提示板专为青少年或成年鱼​​的发展，从而延长了分析可能的范围内。

<html><table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> XF 24外通量分析仪</td><td>海马生命科学</td><td> 100737-101 </td><td> 24孔格式</td></tr><tr><td>胰岛捕获微孔板</td><td>海马生命科学</td><td> 101122-100 </td><td> 24孔格式</td></tr><tr><td> XF校准解决方案</td><td>海马生命科学</td><td> 100840-000 </td><td></td></tr><tr><td> XF检测培养基</td><td>海马生命科学</td><td> 101022-100 </td><td></td></tr><tr><td>油红-O </td><td> Sigma-Aldrich公司</td><td> O0625 </td><td></td></tr><tr><td> 1 - 苯基-2 - 硫脲（PTU） </td><td> Sigma-Aldrich公司</td><td> P7629 </td><td>/ zfbook/chapt10.html＃wptohtml51的"目标="_blank"&gt; http://zfin.org/zf_info/zfbook/chapt10.html＃wptohtml51 </td></tr><tr><td> E3（胚胎介质） </td><td>自制</td><td> - </td><td> <a href="http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml16" target="_blank">http://zfin.org/zf_info/zfbook/chapt10.html＃wptohtml16</a> </td></tr><tr><td> 100X15毫米培养皿</td><td>鹰</td><td> 35-1029 </td><td></td></tr><tr><td> FCCP </td><td>西格玛</td><td> C2920 </td><td></td></tr><tr><td>寡</td><td>西格玛</td><td> 75351 </td><td></td></tr><tr><td>抗霉素A </td><td>西格玛</td><td> A8674 </td><td></td></tr></tbody></table>

metabolic profile analysis of zebrafish embryos

越来越代谢领域的目标是，以确定基因的影响线粒体功能的不同方面。了解这些关系，将有助于了解与线粒体功能障碍，如糖尿病和肥胖相关的疾病的基本病因。仪器仪表的最新进展，使不同的参数的监控线粒体功能的细胞系或组织外植体。在这里，我们提出了一种快速，灵敏的体内线粒体功能的参数分析斑马鱼胚胎发育过程中海马生物科学XF 24外通量分析方法。该协议利用微孔板，每孔放置在一个单一的胚胎胰岛捕捉，可以测量生物能学，包括:（i）基础呼吸;基础线粒体呼吸（二）（三）线粒体呼吸，由于ATP成交;（四）线粒体耦合呼吸或公关oton泄漏及（iv）最大呼吸。使用这种方法的胚胎斑马鱼的呼吸参数可以比较野生型和基因改变的胚胎（突变体，基因的过度表达或基因敲除）或操纵药理学。据预测，本协议的传播，为研究人员提供新的工具来分析此相关的脊椎动物动物模型的体内代谢紊乱的遗传基础。

<html>我们有兴趣了解特定基因的代谢，特别是呼吸速率和脂质代谢的作用。因此，我们治疗野生型斑马鱼胚胎从26高倍视野起与一个特定的药理由这些基因编码的酶的抑制剂。在50 hpf时，一半的车辆和抑制剂处理的胚胎进行了分析使用Seahorse的线粒体功能，而剩下的一半固定在4％PFA过夜，4℃，随后与油红O染色的脂质沉积。治疗与特定酶抑制剂导致1〜2倍，增加在基础呼吸（ 图4），这是与增加脂质沉积，特别是在端脑和下耳泡（ 图4B，C）。 pload/4300/4300fig1highres.jpg"/&gt; 图1。化学处理的斑马鱼胚胎的示意图。野生型基因操纵，或药物治疗，斑马鱼的胚胎是背景。 <img src="/files/ftp_upload/4300/4300fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/4300/4300fig2highres.jpg"/> 图2。 XF24胰岛板成立。（A）四口井含有胚胎的介质温度控制（用X标记），其他/包含一个胚胎。 （B）的特写镜头一个（C6）50 HPF野生型胚胎处理的车辆（DMSO）所涵盖的一个小岛拍摄画面（箭头）。 <img src="/files/ftp_upload/4300/4300fig3.jpg" alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/4300/4300fig3highres.jpg"/> 图3。 Respirat50野生型斑马鱼斑马鱼胚胎中的离子参数。（A）对于每个测量，平均20个独立的胚胎。的基础呼吸（平均数:299.7 SD:75.7; SEM:16.9），最大呼吸（平均数:387.4 SD:93.3; SEM:20.9），备用呼吸能力（平均:87.7; SD:72.0​​; SEM:16.1）。 （B）对于每个测量，平均20个人的结果。基础线粒体的呼吸，由于ATP营业额;线粒体的耦合呼吸和非线粒体呼吸的线粒体呼吸。山:线粒体; SRC:备用呼吸容量SD:标准差），扫描电镜（SEM平均数的标准误差。结果消耗O 2为pmol /分钟/胚胎。 <a href="http://www.jove.com/files/ftp_upload/4300/4300fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/4300/4300fig4.jpg" alt="图4" /> 图4。只代表了sentative结果的基础呼吸和脂质沉积的化学品接触。（A，B）50斑马鱼胚胎油红O染色，在横向视图。胚胎培养的选择性抑制剂（B）显示出更多的油红O染色相比，车辆处理的对照组胚胎前脑（箭头）和下耳泡（箭头）。 （C）基础呼吸〜2倍，增加化学处理的胚胎组（n = 10每个组的胚胎）。 O 2 /最小/胚胎pmol的结果。

Watch this Scientific Journal Video about Metabolic Profile Analysis of Zebrafish Embryos at JoVE.com

Metabolic Profile Analysis of Zebrafish Embryos

斑马鱼代表一个功能强大的的脊椎动物模型，一直用于代谢的研究。在这里，我们描述了一个快速的方法来衡量<em在体内</em&gt;代谢的档案显影斑马鱼，允许不同的线粒体基因或其药理学上操纵的胚胎之间的函数的参数的比较，从而增加这种微生物的适用性。

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

metabolic-profile-analysis-of-zebrafish-embryos

Research

JoVE Journal

Biology

19.8K 次观看.  School of Medicine, Deakin University. 斑马鱼代表一个功能强大的的脊椎动物模型，一直用于代谢的研究。在这里，我们描述了一个快速的方法来衡量

视频: Metabolic Profile Analysis of Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...