Name: construction of an affordable and easy-to-build zebrafish facility
Uploaded: 2014-11-22T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Construction of an Affordable and Easy-to-Build Zebrafish Facility at JoVE.com

笔者想感谢药长老会学院派研究办公室本项目通过小药店奖研究与协作（SPARC）奖的资金支持。作者感谢约翰·Smink先生，水产动物研究​​实验室经理，克莱姆森大学，克莱姆森，SC为他的技术援助的手稿版本。

注意:这些协议遵循长老会学院的IACUC的指导方针。 1.获取的材料列于表1。 2.装配5架镀铬根据制造商的说明。这将用于安置该基金。 3.构建储水器和过滤系统（图1A和2）。 <ol><li>使用图1A作为指导，布局18加仑桶，泵，过滤器和紫外线灯，以确定最后的安装位置和大小。 </li><li>一旦零件摆出来，测量和切¾"PVC管组装所需的长度。根据特定的空间的需要和限制变化所有管道的长度。 </li><li>组装管材和管件的使用PVC底漆和水泥奠定了前面，见下文（3.3.2和3.3.3）。 <ol><li>砂用砂纸管子的切割边缘，直到它们光滑的触感的ð没有碎屑。 </li><li>素管子的接头的与所购买的聚氯乙烯底漆与来自聚氯乙烯底漆包含的拭子的内侧到外侧的和。 </li><li>通过使用附带的拭子施加聚氯乙烯水泥管的外水泥的管材和管件在一起。重复需要PVC固井的所有步骤。 </li><li>放置18加仑桶到搁板结构的一侧。 </li><li>在储槽的侧面安装一个舱壁连接器。 </li><li>附加¾"PVC管的舱壁上。 </li><li>附加¾"管子用3/4泵入口"的另一端部螺纹使嵌合。 </li><li>过滤器连接到使用两个90度弯头和¾"PVC管的泵。 <ol><li>为了在水中过滤掉的碎片和减少氯和化学水平使用一个纸过滤后跟一个活性碳过滤器。 </li></ol></li><li>紫外灯连接到过滤器according到制造商的说明。 </li></ol></li><li>附泵和过滤系统使用线扎机架的第一搁板的顶侧。根据货架/齿条选定的尺寸和布局变化的组装和安装的顺序。 </li><li>添加每加仑水1 biobal​​l到水槽一旦泵和过滤器系统被组装并连接到所述搁板。 </li></ol> 4.构建主供应管线（图1B和2）。 <ol><li>测量切一块¾"PVC管紫外灯连接到主电源</li><li>使用3/4"的PVC三通通过三通连接到¾启动主供应管线"PVC排气从UV滤波器的到来。 。 <ol><li>加入此开闭阀在PVC三通管的底部，作为该系统的维护冲洗漏极。 </li><li> "通过测量和切削¾聚氯乙烯三通"PVC开始从¾顶部的主供应管线管至所需的长度，以提供进料管为每个机架上的架子。 </li><li>安装¾"聚氯乙烯三通为水平线，将进入所述机架的每个货架。 </li><li>任选地（和建议的），开闭阀添加3/4"为每个货架供应管线，以允许针对每个货架的隔离和该系统的模块化设计。 </li></ol></li><li>使用3/4"PVC管作为减压在顶架线。如果该行上的货架的后面运行时，使用该搁板用于存储。结束减压线在主排气管线（ 图1B）与3/4"开-关阀。 </li><li>一旦上述步骤已被完成时，首要和水泥的PVC管一起作为先前构建的贮存器和过滤系统时完成。 </li></ol> 5.构建主排气管线（图1C和2）。 <ol><li>运行主排气管线的垂直长度在机架f的另一侧ROM中的主供应管线。使用2"PVC管这种垂直线以接收主供应管线在从每个货架鱼缸的顶部和漏极线。 </li><li>切的2件"PVC管的垂直主排气管线，并使用2"PVC三通以接收来自每个货架鱼缸漏极线。结束在股票箱的主排气线驱逐循环水通过海绵过滤器。 </li><li>切割一英寸的开口2"PVC管件的整个长度并连接到垂直主排气线与PVC开杆从鱼缸水平漏极线。 </li><li>帽2"横向排水管线用2"PVC帽。 </li><li> Prime和水泥使用必要的配件（接头，乳头或手肘）的作品。 </li></ol> 6.构建鱼缸（图3）。 <ol><li>与钻，切1 1/8"孔在鱼箱1的背面"的上边缘下方有一个孔锯。 </li><lI&gt;滑动的螺纹¾"PVC雄性适配器（装有＃18 O形环）通过孔（从该储箱的内部向外部）。 </li><li>安装并拧紧一个90度的3/4"螺纹PVC弯头男性适配器创建鱼缸排气管。拧紧螺母举行，以防止斑马鱼会通过排气系统前加一块细网。 </li><li>放置在鱼缸其排气管结束进舱内后面的排出管路的开放空间</li></ol> 7.启动供水系统<ol><li>在几个容器，例如用于储存所述一个，测量水三次包含在系统内的量。让这种水在室温下放置进行调理48小时。 </li><li>加水至该系统，让它流通最少3天。 <ol><li>在系统大力整个循环的最初3天，以消除污染物塔系统换水吨驻留在管道内。 </li></ol></li><li>添加启动斑马鱼，并允许得到确立的氮循环。标准化pH值一致7.保持7斑马鱼每1.8 L，并作为建议林波4和6麦克纳布在周末，每天两次在平日喂它们，每日一次。 </li><li>监视硝酸盐（&lt;75 mg / L时），亚硝酸盐（&lt;10毫克/升），和铵（0毫克/升）每日在第一周期间加入鱼后，和每周一次所需的两倍的水平一直保持定期的换水。 </li></ol>

<ol>
	<li>Detrich, H., Westerfield, M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Cellular and Developmental Biology, Part A.. , (2010).</li><li>Grim, J. 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=Hemogen+(hemgn),+a+transcription+factor+that+regulates+erythropoiesis+and+skeletogenesis+in+zebrafish.">Hemogen (hemgn), a transcription factor that regulates erythropoiesis and skeletogenesis in zebrafish.</a> , (2013).</li><li>Kim, S., Carlson, R., Zafreen, L., Rajpurohit, S. K., Jagadeeswaran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular,+easy-to-assemble,+low-cost+facility.">Modular, easy-to-assemble, low-cost facility.</a> Zebrafish. 6 (3), 269-274 (2009).</li><li>Linbo, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+husbandry+and+colony+maintenance+at+the+Northwest+Fisheries+Science+Center.">Zebrafish (Danio rerio) husbandry and colony maintenance at the Northwest Fisheries Science Center.</a> U.S. Dept. Commer., NOAA Tech. Memo. 62, (2009).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. 69, 4196 (2012).</li><li>McNabb, A., Scott, K., Ochsenstein, E., Seufert, K., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+be+afraid+to+set+up+your+fish+facility.">Don't be afraid to set up your fish facility.</a> Zebrafish. 9 (3), 120-125 (2012).</li><li>Garcia, R. L., Sanders, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+cleaning+and+disinfection+procedures+in+a+zebrafish+rerio)+facility.">Efficacy of cleaning and disinfection procedures in a zebrafish rerio) facility.</a> J Am Assoc Lab Anim Sci. 50 (6), 895-900 (2011).</li><li>Rivas-Boyero, A. 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=Pharmacological+characterization+of+a+nociceptin+receptor+from+zebrafish+(Danio+rerio).">Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).</a> J Mol Endocrinol. 46 (2), 111-123 (2011).</li><li>Sanchez-Simon, F. M., Zhang, X. X., Loh, H. H., Law, P. Y., Rodriguez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+regulates+dopaminergic+neuron+differentiation+via+miR-133b.">Morphine regulates dopaminergic neuron differentiation via miR-133b.</a> Mol Pharmacol. 78 (5), 935-942 (2010).</li><li>Velasco, E. M., Law, P. Y., Rodr&#237;guez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mu+opioid+from+the+zebrafish+exhibits+functional+characteristics+as+those+of+mammalian+mu+opioid+receptor.">Mu opioid from the zebrafish exhibits functional characteristics as those of mammalian mu opioid receptor.</a> Zebrafish. 6 (3), 259-268 (2009).</li></ol>

作者宣称对本文没有利益冲突。

本文的主要目的是要证明的功能斑马鱼设施建设不复杂，并且可以通过新手鱼的研究人员以最小的结构的知识来构建。此外，这篇文章显示，对于这一功能的斑马鱼设施的材料可从当地的宠物用品商店或硬件商店容易，经济地获得。一旦所有的材料和工具已经获得了系统的建设和鱼缸大约需要1个工作日。在72小时的第一次运行可以在系统中的任何故障，例如泄漏或低水压，待确定。这第一次运行?...

体内模型是进行全面的生物医学研究，因为它们允许在体外研究的系统和有机体水平翻译是必不可少的。例如，对于小的哺乳动物，即啮齿动物，研究已经证明有必要在药物的开发和在可用药物的提高。然而，复杂性（财政和后勤），可能使其几乎不可能建立和维护设施的啮齿动物做实验，提出在小型研究的学术机构。然而， 在体内研究可以使用需要更少精细物流其它动物物种中进行。斑马鱼（ 斑马鱼 ）已经被成功地用作在体内模型来研究遗传学，发展，病理生理学，药理学和1,2,10,11,12。相比于r容纳斑马鱼进行实验所需的设施都比较简单和显著便宜odent设施。 Kim 等人 3已经详细描述了最近的80罐的斑马鱼壳体系统，它可以为约1500，其包括两个部分和劳动力成本来构建。此形成鲜明对比的是近8500美元将需要从任意数量的商业供应商购买一个现成的，80箱系统。然而，由Kim 等人给出的描述。可能是复杂和难以遵循鱼研究的新手。我们认为，斑马鱼设施可以简化的方式为1500美元的投资来构建。 体内设备的小型学术机构的建立提供了自己的才能，用以生产更强大，更深入的研究一个额外的工具， 在体内的结果支持在体外的数据通常会产生更全面的生物医学研究比仅采用体外方法获得的数据。因此，该实现的体内研究设施将显著提高该机构的成功在其总体研究工作（学生接触到研究，出版，通讯，追求外部资金等 ）。 的斑马鱼设施像在这项研究中所描述的建设是收缩金融时报在理想的科研学术机构与设施有限（或机构没有在体内研究设施）。

<table><tbody><tr><td>Item</td><td>Brand</td><td>Quantity</td><td></td></tr><tr><td>74-in H x 48-in W x 18-in D 5-shelf Steel Freestanding Shelving Unit</td><td>Style Selections</td><td>1</td><td></td></tr><tr><td>18-Gallon General Bucket/Tote</td><td>Centrex Plastics: LLC&amp;nbsp;Rugged Tote</td><td>1</td><td></td></tr><tr><td>6-Outlet Power Strip With 15-Ft. Cord</td><td>Master Electrician</td><td>1</td><td></td></tr><tr><td>9-Watt UV Water Garden Clarifier FiIter and UV lamp</td><td>Smartpond&amp;nbsp;</td><td>1</td><td></td></tr><tr><td>Whole House Water Replacement Filter</td><td>Whirlpool</td><td>6</td><td></td></tr><tr><td>Opaque Whole-House Pre-Filtration Housing</td><td>Whirlpool</td><td>2</td><td></td></tr><tr><td>Thread Seal Tape</td><td>William H Harvey</td><td>1</td><td></td></tr><tr><td>8-fl oz LO-VOC PVC Cement</td><td>Oatey&amp;nbsp;</td><td>1</td><td></td></tr><tr><td>8-fl oz LO-VOC Primer</td><td>Oatey&amp;nbsp;</td><td>1</td><td></td></tr><tr><td>1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing</td><td>Mister Landscaper&amp;nbsp;</td><td>1</td><td></td></tr><tr><td>1/4-in Barbed Drip Irrigation On/Off Valve</td><td>Mister Landscaper</td><td>33</td><td></td></tr><tr><td>9-Port NPT Irrigation Manifold with Filter</td><td>&amp;nbsp;Mister Landscaper</td><td>6</td><td></td></tr><tr><td>Drop-In Filter with House&amp;nbsp;</td><td>Whirlpool</td><td>6</td><td></td></tr><tr><td>2-in Dia 90-Degree PVC Elbow</td><td>LASCO</td><td>3</td><td></td></tr><tr><td>2-in Dia 90-Degree Tee</td><td>LASCO</td><td>3</td><td></td></tr><tr><td>3/4-in Dia 90-Degree Slip Elbow</td><td>LASCO</td><td>15</td><td></td></tr><tr><td>3/4-in Dia PVC Adapter</td><td>LASCO</td><td>7</td><td></td></tr><tr><td>3/4-in Dia PVC Tee</td><td>LASCO</td><td>12</td><td></td></tr><tr><td>2-in Dia PVC Cap</td><td>LASCO</td><td>3</td><td></td></tr><tr><td>1-in Dia x 3/4-in Dia PVC Adapter</td><td>LASCO</td><td>2</td><td></td></tr><tr><td>3/4-in PVC Socket In-Line Ball Valve</td><td>American Valve</td><td>5</td><td></td></tr><tr><td>1/2-in Dia x 3/4-in Dia PVC Adapter</td><td>Lasco</td><td>8</td><td></td></tr><tr><td>3/4-in Dia PVC Cap</td><td>Lasco</td><td>1</td><td></td></tr><tr><td>2-In x 5-Ft Pvc Pipe</td><td>Charlotte Pipe</td><td>4</td><td></td></tr><tr><td>&amp;frac34;-In x 10-Ft Pvc pipe</td><td>Charlotte Pipe</td><td>3</td><td></td></tr><tr><td>11/16x1-7/80 PVC ring</td><td>SXT</td><td>10</td><td></td></tr><tr><td>&amp;frac12;&amp;rdquo; PVC male adapter</td><td>SXT</td><td>1</td><td></td></tr><tr><td>&amp;frac34;&amp;rdquo; PVC coupling&amp;nbsp;</td><td>SXT</td><td>1</td><td></td></tr><tr><td>&amp;frac12;&amp;rdquo; PVC female adapter&amp;nbsp;</td><td>SXT</td><td>1</td><td></td></tr><tr><td>link</td><td></td><td></td><td></td></tr><tr><td>1.8 L Dual Beta Keeper</td><td>Top Fin</td><td>27</td><td></td></tr><tr><td>&amp;frac34;&amp;rdquo; PVC male adapter (w/ #18 O-ring)</td><td>SXT</td><td>28</td><td></td></tr><tr><td>90-degree &amp;frac34;&amp;rdquo; threaded PVC elbow</td><td>SXT</td><td>27</td><td></td></tr><tr><td>Fine Mesh</td><td>N/A</td><td>1 yard</td><td></td></tr><tr><td>Bulkhead Connector</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Hacksaw</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Drill</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Hole saw</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Adjustable Wrench</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Pliers&amp;nbsp;</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Tape Measure</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Safety Glasses</td><td>N/A</td><td>1</td><td></td></tr><tr><td>Work Gloves</td><td>N/A</td><td>1</td><td></td></tr></tbody></table>

construction of an affordable and easy-to-build zebrafish facility

在体内的生物医学研究的关键是在体外成果转化为临床应用进展翻译。小型学术机构以有限的资源发现它几乎是不可能的建立和维护典型的啮齿动物的设施进行研究。斑马鱼的研究已经证明是用于体内研究的药理学，生理学，发展和遗传研究一个有价值的替代。本文演示了一个功能斑马鱼设施可以建在一个简单而实惠的方式。我们证明，这样的设施可以建在大约一个工作日以最小的工具和专业知识。本研究构建了27 1.8升鱼缸斑马鱼设施的成本约为1500美元。我们估计，维修的初始工作150鱼殖民地的3个月是1000美元。这涉及到项目的学生，谁被介绍给aquaculturing斑马鱼的研究提出。

泵和Flters体系建设 一旦机架组装和设置的第一步是建立泵和过滤系统。该过滤器被连接到该系统的泵和位于上方的第一搁架，其允许容易过滤器维护和更换的底部水平。类似地，UV灯与PVC管的过滤器连接和代替用塑料丝的关系保持，以允许容易地更换紫外灯的需要（ 图4A）。紧接纸过滤，活性碳过滤器，和UV灯通过PVC管安装在泵上。 Biobal​​ls被添加到罐中细菌的生长，?...

Watch this Scientific Journal Video about Construction of an Affordable and Easy-to-Build Zebrafish Facility at JoVE.com

Construction of an Affordable and Easy-to-Build Zebrafish Facility

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

一个经济实惠，易于构建斑马鱼设施建设

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

construction-of-an-affordable-and-easy-to-build-zebrafish-facility

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.3K 次观看.  Presbyterian College School of Pharmacy. A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

视频: Construction of an Affordable and Easy-to-Build Zebrafish Facility

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

科研

环境科学

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

发育生物学

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

神经科学

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

研究斑马鱼光遗传学信号转导操作的发育

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

作者聚焦：操纵斑马鱼胚胎中的信号转导以解码细胞命运决定 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

斑马鱼胚胎中的光遗传学信号激活

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

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 ...

生物学

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

一个经济实惠，易于构建斑马鱼设施建设

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此视频中的章节

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

斑马鱼幼虫显微注射和成像的微装置

在斑马鱼幼虫的高通量自动化的行为分析

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

定期保养和维护的斑马鱼（

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

手册小分子屏幕接近高通量使用斑马鱼胚胎