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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;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&#160;</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&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC Primer</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing</td>
			<td>Mister Landscaper&#160;</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>&#160;Mister Landscaper</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Drop-In Filter with House&#160;</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>&#190;-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>&#189;&#8221; PVC male adapter</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC coupling&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC female adapter&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>link</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>&#190;&#8221; PVC male adapter (w/ #18 O-ring)</td>
			<td>SXT</td>
			<td>28</td>
			<td></td>
		</tr>
		<tr>
			<td>90-degree &#190;&#8221; 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&#160;</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

Research

JoVE Journal

Biology

16.2K 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

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

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 ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...