作者想感谢斑马鱼研究人员对他们的关心和维护的斑马鱼的Freimann生命科学中心和中心。

1。重悬吗啉<ol><li>到100μL无核酸酶水稀释300纳米荧光标记啉，使约3毫米的解决方案。吗啉溶液分装成多个石蜡密封离心管中，并在室温和避光储存。 </li><li>以确定确切的吗啉浓度，稀释在95μL的0.1 N的盐酸吗啉溶液5μL（或水）作为空白。设置上与水的空白，在265 nm的分光光度计基线，然后确定稀释的吗啉溶液的读数。待定常数乘以稀释倍数啉吸收，以确定在ng /μl的浓度。啉常数的计算公式为： 吗啉常数=啉X 1000/molar吸收的分子量。 吗啉的分子量和摩尔吸光可以发现“寡属性“表提供的产品。除以啉浓度，ng /μl的分子量确定以毫米为单位的浓度，稀释吗啉，如有必要，工作浓度，一般为1.2毫米。 </li></ol> 2。鳍截肢<ol><li>麻醉成年斑马鱼在，要么Tricaine或2 - 苯氧乙醇在1.0毫克/毫升水箱。 </li><li>使用无菌手术刀或刀片第一lepidotrichial分支点近端截肢的鳍。这项工作应在相同的近端/远端对每个动物的位置（例如7鳍腰带从远端的骨段）。要点：确保削减完美动物的前/后平面垂直。角度的削减将导致不平衡鳍的鳍背，腹部分生长。 </li><li>鱼坦克。我们通常鱼保持在33°C到提高再生率。根据实验设计，WAIT 0-2天截肢后翅再生（DPA）开始前引进的吗啉。替代方法在下面的讨论，讨论。 </li></ol> 3。吗啉注射液<ol><li>前一天注射吗啉，使注塑板（ 图2）。确保切出一个缺口，在一端的好，这有助于稳定的显微注射过程中的鱼。 </li><li> 2 DPA，准备微注射器具及吗啉的解决方案。 </li><li>稀释的荧光标记啉适当浓度（推荐1.2毫米起）和地方在65℃水浴5分钟。 </li><li>使用微量注射一针拔拉玻璃针。 </li><li>加载啉针（注：无论你回来填充或前加载你的针，将决定你是否加载第一针，或削减尖端而定）。 </li><li>在削减了针的尖端ñ角。 </li><li>按照您的微喷射系统的方向，注入约5 NL啉的泡沫，每次注射。啉量较大，可能会破坏组织。 </li><li>麻醉注射板鱼和地方。删除所有多余的液体和东方的针到适当的位置，只是远端骨线（ 图3）。使用显微镜和显微注射仪适合吗啉注射液（见表的特定试剂），由前至后翅注入吗啉， 如图3所示。从后至前注射会导致鳍组织在注射推出，是非常困难的。 </li><li>针轻轻插入到再生的组织，只是远端每个骨射线（标有“1”，在图3）和远端推，直到位于胚（标有“2”，在图3）。要小心，不要推针通过EN轮胎的鳍。当针正确定位，注入吗啉（即点击一次）。在1.2毫米，出现荧光标记的吗啉溶液的黄绿色，即使在正常光线条件下。每次注射，一个黄色的“噗”吗啉溶液可以可视化，这有助于本地化注射到胚。约75 NL（10-15点击）吗啉溶液应注射每骨射线。暂停〜1秒之间人次。我们只注入背侧，使用电，只有控制腹。另外，可以重复使用这一整个过程控制啉翅腹面的一半。 </li></ol> 4。电，对吗啉<ol><li>紧随注射吗啉，从微注射器具注射板。填写麻醉液注射板鱼，直到被淹没。水位线下的电。 </li> &lt;李&gt; 3毫米直径的铂金板钳电极（CUY 650-P3镊子，保得国际）定位脉冲约一个半翅。小心不要触摸电极鳍组织。为了确保电极不接触鳍组织，在其背侧旋转的鱼和直视下电中线。 </li><li> Electroporate都背，腹（控制一个非特定的电效应）使用1 CUY21广场，波Electroporator（保得国际公司）双方的鳍。擦拭后，每个电潮湿KimWipe，以消除任何可能形成的泡沫电极。如果他们不删除，迷你负责建立，这将吸引组织向电极。应设置连续十50毫秒脉冲的电参数，在15至五1秒脉冲之间的停顿。 </li><li>将玻片或培养皿和快速的图像鳍鱼，确保到n大手，背/腹方向。翌日，该图像将被用于再生分析。鱼返回到坦克。 </li></ol> 5。分析<ol><li>如果目标蛋白表达下调被撞倒在适当的鳍再生，显着的差异之间的鳍背，腹部分应该是很明显的后1天，电（ 图5B）。 </li><li>在3 DPA，另取每条鱼的鳍的图片，并匹配相应的2 DPA（ 图5B）这个图片。在动物之间的鳍色素沉着模式的自然变化将使你正确识别个别鱼。 </li><li>使用NIH图像，追踪2的背部和腹部的一半（ 图5B）政治部和3 DPA领域。 </li><li>要确定面积背侧与腹侧使用下列公式：％（背3dpa -背2dpa的 ）/（腹3dpa -<html>腹侧2dpa）×100 =％面积</li></ol> 6。代表结果<ol><li>我们始终检测鳍生长在3 DPA，或24 HPE（小时后电）。荧光标记的吗啉在这个时候，应该在目前的鳍背一半（ 参考图1和图6A）。看到一些尾随截肢的水平，这是正常的。 </li><li>与控制吗啉代（ 图6B）注射和电穿孔的鳍背，腹两侧之间不应该有差异。 </li><li>如果实验啉目标鳍生长必需的蛋白质，背侧和腹侧双方之间的巨大差异，应遵守（ 图6C）。 </li><li>如果电极在电鳍感动，该组织将出现褐色（在外观上几乎烧毁）和坏死。 </li></ol>“SRC =”/ files/ftp_upload/3632/3632fig1.jpg“/&gt; 图1。鳍再生过程中发生的各种事件的示意图。每个事件的相关时间以小时后截肢（HPA）和对应的水箱温度33°C。 <img alt="图2" src="/files/ftp_upload/3632/3632fig2.jpg" /> 图2。注塑板，这是由琼脂糖和包含一个小以及期间举行的注射吗啉鱼的示意图。 <img alt="图3" src="/files/ftp_upload/3632/3632fig3.jpg" /> 图3。鱼菜头鱼在缺口啉显微注射示意图。A.将切出井，这将有助于鱼保持稳定。B.在低倍率，安排针，使靠近鳍的再生组织。 </stro吴&gt;在更高的放大倍率，注入啉远端各骨的鳍条（即在每个芽）。针应该进入只是远端骨射线（1），组织，然后继续胚（2）的位置。注：在原理图中的绿色圆圈，只是为了显示注射的位置。吗啉绿色/黄色“噗”，每次注射后，可以简单的可视化;然而，这并不存在，如示意图所示。 <img alt="图4" src="/files/ftp_upload/3632/3632fig4.jpg" /> 图4。鳍电原理图。A.显微注射后，放置在培养皿全麻醉的鱼和electroporate背，腹部分。确保不碰鱼翅组织。电极应放在〜1毫米的组织。 <img alt="图5" src="/files/ftp_upload/3632/3632fig5.jpg" /> <strong图5。用来计算鳍生长抑制的方法示意图。A.以政治部，无论之前或之后立即啉注射和电鳍鱼的每个图片。跟踪背（绿色）和腹侧（蓝色），NIH图像（黑色虚线）B.使用3 DPA鳍半再生组织，采取其他各鳍的图片，并再次追踪的背，腹地区再生利用NIH图像。减去2 DPA在3 DPA对于背部和腹部的富人总再生的再生领域。 ％背侧与腹侧重新生长面积，可以计算公式：（（四3dpa - D 2 DPA）/（五3dpa - 2dpa至五））×100。百分之抑制= 100 - ％的面积。 <img alt="图6" src="/files/ftp_upload/3632/3632fig6.jpg" /> 预计图6。结果。A.控制啉荧光素标记的荧光图像显示在鳍背一半，24小时后电（HPE）。明视图像的注射与控制啉电穿孔的鳍背半。图像显示的背部和腹部的鳍，24 HPE半等于再生。背半的实验吗啉，注射和电穿孔翅片C.明视的形象。图像显示背/注射侧抑制再生。行显示的金额在2 DPA，紧接啉注射和电再生。 <img alt="图7" src="/files/ftp_upload/3632/3632fig7.jpg" /> 图7。交替注入和电在伤口愈合和胚形成过程涉及到目标蛋白的示意图。A.注入啉彼此之间骨鳍条上的鳍背一半。Electroporate按正常吗啉。C.截肢鳍立即注射部位近端（〜1骨段）。荧光标记啉可以观察在伤口上皮细胞和胚在24 HPE。 <img alt="图8" src="/files/ftp_upload/3632/3632fig8.jpg" /> B.图8。使用的技术为目标伤口上皮细胞和胚形成。明视在24百帕鳍控制啉注射，电穿孔立即截肢前的形象。鳍的荧光图像显示在面板A.注意注入一半的鳍背，显示了良好的再生组织中摄取的吗啉。 C - C“的高倍在面板中显示的鳍背一半，A和B注射部位往往仍清晰可见（箭头）但许多有针对性的细胞迁移参与创面上皮细胞和胚形成（箭头）。 <img alt="图9" src="/files/ftp_upload/3632/3632fig9.jpg" /> 图9。利用这一技术来针对细胞迁移到形成胚。A.荧光灯和明显示鳍注入，背，腹部分啉电穿孔的插图图像。在两架飞机的鳍，然后截肢。背半立即被切断，远端注射部位，那里的腹侧被削减一半9-10远端注射部位的骨段。B.在24百帕，荧光灯和明插图图像表明，吗啉已迁移到背再生（1），而不是腹重生（2），这表明仅在细胞立即截止站点近端参加原基形成。两套白色箭头显示每个截肢飞机。板上标明的最右边的图像显示更高的放大倍率视图的鳍背，腹部分，分别为1和2。 <a href="http://www.jove.com/files/ftp_upload/3632/Suppl_Fig1_JoVE.mov">参考图1。</a>一个视频共聚焦Z-Stack的一个区域对应的胚在鳍控制啉注射和电穿孔的位置。 24 HPE图像。由于单个荧光分子不能显示，不是所有的吗啉可以可视化或量化。然而，这些图片给一些吸收不同程度的细胞，从单个点状点，吗啉荧光充满整个细胞，可以在可视化的想法。在静止图像上，方向显示。比例尺：25微米。

<ol>
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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, 216-2120 (2000).</li><li>Hoptak-Solga, A. D., Nielsen, S., Jain, I., Thummel, R., Hyde, D. R., Iovine, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin43+(GJA1)+is+required+in+the+population+of+dividing+cells+during+fin+regeneration.">Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration.</a> Dev. Biol. 317, 541-548 (2008).</li><li>Thummel, R., Bai, S., Sarras, M. P., Song, P., McDermott, J., Brewer, J., Perry, M., Zhang, X., Hyde, D. R., Godwin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+zebrafish+fin+regeneration+using+in+vivo+electroporation+of+morpholinos+against+fgfr1+and+msxb.">Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.</a> Dev. Dyn. 235, 336-346 (2006).</li><li>Yin, V. P., Thomson, J. M., Thummel, R., Hyde, D. R., Hammond, S. M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf-dependent+depletion+of+microRNA-133+promotes+appendage+regeneration+in+zebrafish.">Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish.</a> Genes Dev. 22, 728-733 (2008).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

我们什么都没有透露。

在这里，我们描述了一个利益有条件击倒蛋白质功能强大的亏损在成年斑马鱼的鳍再生的方法。这项技术已用于研究间隙连接蛋白基因，信号受体，转录因子，小分子RNA在再生鳍的产物16，20-22。 我们预计，这种技术也可以用来研究基因伤口愈合和原基形成需要适应技术。例如，我们注入和控制啉之间的骨鳍条上的鳍背一半的空间电穿孔前截肢（ 图7）。然后，我们立即注射平面远端截肢的鳍。 24百帕，我们观察到啉靶细胞迁移远端形成的伤口上皮细胞和胚（ 图8），表明在这些早期阶段的再生细胞也可以TARG的eted。 该技术具有几个显着的局限性。例如，从荧光标记的荧光不坚持免疫组化，这使得它不可能关联啉金额在细胞中存在一个特定的细胞表型（即细胞增殖）以下的固定和处理。此外，我们已经无法坚持实现再生尾鳍质粒电，虽然前一组报告成功的DNA电成鳍组织23。最后，我们注意到，吗啉是只〜48小时后电21日 ，禁止使用这种技术，在其目前的形式参与新类型的细胞分化的基因测试有效。额外的测试和修改程序，可能会克服目前的局限性。 此外，它是可能的，这种技术可以用来测试从底层组织的胚细胞迁移的蛋白质参与。例如，我们分为两个鳍两侧（ 如图7），截肢前控制啉注射和电穿孔。然后，我们截肢鳍立即注射平面远端背侧半，我们被截肢的腹鳍更远端。在24百帕，背侧啉阳性细胞已迁移从注射部位覆盖创面上皮细胞及胚。然而，这不是腹侧方的情况（ 图9）。这支持的想法，只有截肢平面的基础细胞参与的再生反应。这些数据还表明，假设为细胞迁移所需的蛋白质，可以使用这种技术进行测试。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> CUY21-EDIT或CUY21-SC方波electroporator的</td><td>保得国际</td><td> CUY21EDIT或CUY21SC </td><td>这两个单位的工作此协议</td></tr><tr><td>直径3毫米桨电极</td><td>保得国际</td><td> CUY 650-P3的</td><td></td></tr><tr><td>吗啉</td><td> GeneTools，LLC </td><td></td><td>吗啉应该可以定制您感兴趣的蛋白质</td></tr><tr><td> 2 - 苯氧乙醇</td><td>西格玛</td><td> 77861-1L </td><td>麻醉;稀1:1000鱼制水过程中，安乐死1:500 </td></tr><tr><td>微-injecti在泵</td><td>世界精密仪器</td><td> PV830气动PicoPump </td><td>可用于许多不同的显微注射系统</td></tr><tr><td>微型机械手</td><td>世界精密仪器</td><td> MMJR </td><td>右手（左手MMJL） </td></tr><tr><td>微注射针头，外径1.0毫米</td><td>世界精密仪器</td><td> 1B100F-4 </td><td>这些都是高硼硅玻璃毛细管，拉成针</td></tr><tr><td>持针器</td><td>世界精密仪器</td><td> 5430 - ALL </td><td>微型喷嘴套件;确保插入1.0毫米的吸管垫片</td></tr><tr><td>针拔</td><td>萨特</td><td>的P-97 </td><td>其他微量/针车夫也应努力</td></tr><tr><td>显微镜</td><td>莱卡，尼康，Zeis小号</td><td>根据制造商数目不同</td><td>任何20X光学体视显微镜和工作能力与显微操作</td></tr></tbody></table>

in vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin

某些物种的有尾类和硬骨鱼可以再生自己的组织。斑马鱼已成为一种广泛使用的模型来研究成人的心脏，视网膜，脊髓3，视神经4，感觉毛细胞，鳍6，组织，如自发再生。 斑马鱼的鳍是一种相对简单的附属物，很容易被操纵到研究在epimorphic再生多个阶段。经典，鳍再生的特点是由三个不同的阶段：创面愈合，芽基形成，和鳍的产物。后截去鱼翅的一部分，周围的上皮细胞增殖和迁移的伤口。在33°C时，此过程中发生截肢后的六小时内（HPA， 图1B）6,7。其次，从不同的谱系（如骨骼，血液，神经胶质细胞，成纤维细胞）的基本细胞重新进入细胞周期，以形成一个增殖芽，W类型改为覆表皮细胞继续增殖（ 图1D）8。生长发生的胚细胞近端重新纳入各自的谱系分化形成新的组织（ 图1E）8。根据截肢的水平，充分再生完成后，在一个星期到一个月。 大量的基因家族，包括WNT，Hox基因，FGF，MSX，维甲酸，SHH，缺口，BMP，和激活素-β的一个基因，表达上调的鳍再生9-16期间的特定阶段。然而，这些基因编码的蛋白质和再生过程中的角色已经难以评估，除非特定蛋白质抑制剂存在13，温度敏感突变体的存在，或转基因动物（无论是过度的野生型蛋白或占主导地位负蛋白）产生的7,12。我们develoPED反向遗传技术，快速，方便地测试鳍再生过程中的任何基因的功能。 吗啉代寡核苷酸被广泛用于研究斑马鱼， 爪蟾 ，鸡，和鼠标的发展17-19期间的特定蛋白质的损失。吗啉代碱基互补的RNA序列，要么块前体mRNA剪接或mRNA的翻译。我们描述了一种方法，能够有效地引入到斑马鱼鳍再生击倒的目标蛋白表达的荧光标记的反义吗啉代。吗啉是微注射到每个芽再生斑马鱼尾鳍，并把周围的细胞电穿孔。荧光提供负责electroporate啉和形象化吗啉在翅片组织的。 该协议允许有条件的蛋白质击倒审查再生鳍生长过程中的特定蛋白质的作用。在探讨TheN，我们描述了这种方法可以如何适应研究特定蛋白质的作用，在伤口愈合或原基形成，以及胚形成过程中的细胞迁移的潜在标记。

Watch this Scientific Journal Video about In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin at JoVE.com

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

我们描述了一个方法，有条件击倒目标蛋白的表达，在成年斑马鱼的鳍再生。这项技术涉及微注射和反义寡核苷酸进入鳍组织，它允许测试鳍再生，伤口愈合，芽基形成和再生产物，包括在不同阶段的蛋白质的作用吗啉代电穿孔。

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 ...

in-vivo-electroporation-morpholinos-into-regenerating-adult-zebrafish

Research

JoVE Journal

Biology

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

16.8K 次观看.  University of Notre Dame. 我们描述了一个方法，有条件击倒目标蛋白的表达，在成年斑马鱼的鳍再生。这项技术涉及微注射和反义寡核苷酸进入鳍组织，它允许测试鳍再生，伤口愈合，芽基形成和再生产物，包括在不同阶段的蛋白质的作用吗啉代电穿孔。

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...