作者想感谢的关心和维护斑马鱼斑马鱼研究人员Freimann生命科学中心和中心。

1。吗啉的准备: <ol><li>所有的吗啉代寡核苷酸应定制设计，并通过GeneTools有序（Philomath，OR）。我们使用的里沙明标签带正电的morpholinos驱动实现负电极的电穿孔morpholinos。我们尝试过，但没有成功，目标带负电荷的荧光标记morpholinos的正极，使用这种技术。 100μL无核酸酶水稀释成300 nmol啉产量3毫米的解决方案。分装成多个石蜡密封离心管吗啉。在室温下避光储存。 </li><li>确定稀释5μL啉（或用来稀释为空白啉的水），在95μL的0.1 N的盐酸吗啉浓度。设置分光光度计波长（λ）为265 nm。计算使用下列公式啉的常数: 常数=分子量特别是你吗啉O X 1000/molar吸光度。 </li><li>你会发现"寡糖属性"表提供每个啉的分子量和摩尔吸光度数据。分析的空白，以验证其基准读数和每个稀释啉读数。其待定常数，稀释倍数乘以每个啉在265 nm的吸光度，以ng /μL的浓度。这个数字除以分子量确定以毫米为单位的浓度。稀释的吗啉，如果必要的话，一个工作浓度。 </li></ol> 2。卸下外层角膜层: <ol><li>麻醉6-12个月Tricaine或1.0毫克/毫升，斑马鱼的鱼缸水2 - 苯氧乙醇岁成年斑马鱼。 </li><li>包装在一个斑马鱼蘸纸毛巾，覆盖鳃，但眼睛暴露。包装的鱼被放置在一个立体的放大倍数增加，直到填充眼的直径大约三分之一的视野（图1）。 </li><li>使用杜蒙＃5镊子，抢附近的视神经裂外层角膜。这个组织被染成红色，并标有"业主立案法团"，在图1。有轻微的突起或标有"瘦肉精"的箭头所指示的唇。在整个眼低角度（即10度），拉出外层角膜。随着实践中，这可以完成一项议案。如果镊子滑，重抓组织，并删除之前，再次以低角度拉。一个附加的可引起角膜的眼睛被拔出时，从插座脱落。您可以继续成为steadying另一套镊子脱落的眼睛。 </li></ol> 3。角膜切口和吗啉注射: <ol><li>紧随去除外层的角膜，使角膜小切口瞳孔与虹膜（图2）。这是使用蓝宝石手术刀刀片（见材料）。 </li><li>装入一个Hamilt 0.5微升3.0毫米啉解决方案配备了33号可拆卸，钝针（见材料）上的注射器。移液器上下线，以消除气泡。通过切口（图2），小心地插入玻璃体针。不要太远，否则您将穿刺视网膜的针插入或置换镜头。 </li><li>慢慢地注入vitreal空间吗啉的解决方案。根据鱼的年龄，vitreal空间将于〜0.5μL。你应该想象本色切口玻璃体泄漏，因为它是由啉溶液流离失所。直到啉解决方案泄漏的少量注入了切口。你应该清楚地可视化里沙明标签啉眼内。 </li><li>注射后，返回鱼坦克恢复。我们通常只注入左眼，使用右眼作为uninjected对照，但双眼电击是有可能的。 </li></ol> 4。电穿孔: <ol><li>以下注入约10条鱼，重新麻醉注射鱼和包裹在纸巾浸湿一块，正好覆盖鳃，但眼睛暴露。鱼转移到培养皿中。乳胶或类似的手套，以避免暴露于麻醉。轻轻地捧着鱼盘底部，填写麻醉的菜（图3，左图）。 </li><li>一个直径3毫米的铂金板电极（见材料）本地化的电脉冲视网膜的约一半。我们通常的目标背视网膜。轻轻向下按正极上的眼睛腹侧一半。删除与外角膜，眼睛很容易旋转，暴露眼球的背部分。 </li><li>尽管仍然按眼腹侧，将附近的负电极（〜1 - 2毫米）暴露的眼睛背一半。小心不要触摸负电极直接到眼睛，这将导致损坏背视网膜。一旦你确定适当的电极之间的距离，使用电极的手柄调节螺丝，保持传播时，电穿孔。这使负极从按下位置时，正极的眼球的最佳距离。 </li><li> Electroporate使用一个CUY21方波Electroporator（见材料）的眼睛。电参数应设置为两个连续的50毫秒脉冲，在75 V之间的脉冲1秒暂停。 </li><li>鱼返回油箱。我们通常恒定的光致视网膜变性后立即电击开始我们的协议。 </li></ol> 5。代表性的成果: <ol><li> 3-5天以下的电（取决于啉的稳定），里沙明标签可在切片视网膜视网膜层（图4）可视化。这作为一个在场的确认标记吗啉。 </li><li>我们可以实现完整的蛋白质击倒以下电击可达3-5天，这取决于啉的疗效（图5）。通常情况下，在光照治疗研究中，我们收获吗啉电穿孔后3或4天的眼睛。这是由弧形的两端（图6，材料）与杜蒙＃5B钳取出整个眼球和组织处理 15免疫组化。任何敲除技术，高效击倒示范必须证明为了验证表型。因此，我们建议您感兴趣的蛋白质获得抗血清。另外，如果和抗体不和吗啉是向任前体mRNA的剪接受体或供区，它会阻止有效的mRNA剪接。在这种情况下，可以使用逆转录PCR扩增在morphant RNA的侧翼序列，并确定阻断正常splic的工作效率ING模式。 </li><li>当视网膜切片从鼻腔背腹轴的时间，蓝框的阴影（图6）代表的是最常见的电击事件本身损坏的视网膜的领域。阴影粉红色的框代表啉引入成视网膜细胞电穿孔的目标区域。 </li></ol><img alt="图1。" src="/files/ftp_upload/3603/3603fig1.jpg" /> 图。图1描绘去除角膜上皮细胞层。左侧面板显示眼球的主要结构特点，包括镜头（L），玻璃体（VIT），视网膜（R），外角膜（OC），内角膜（IC），角膜凸出（ CL）眼睛的方向显示在右下角的第一个面板和标记（一）前，后（P），背（四），腹（五）。红色着色表示被删除，外层的角膜。第二届泛<html> EL显示如何使用镊子把握角膜前突和低角度（第三个面板）拉整个眼睛外层角膜。右侧的面板显示的第二双钳可用于稳定的眼睛，同时拉动了角膜。 <img alt="图2。" src="/files/ftp_upload/3603/3603fig2.jpg" /> 图2:在玻璃体内注射的吗啉所涉及的步骤示意图。小切口是在眼睛的瞳孔与虹膜（左侧面板）。这个差距应该足够大，为33号针头，这是充满啉的解决方案，要插入（中间面板）。慢慢地注入玻璃体（右图）0.5μL解决方案。你应该看到一个vitreal解决方案的小切口玻璃体部分取代与里沙明标签吗啉（右图）。 ig3.jpg"/&gt; 图3。一般的电穿孔过程的示意图。麻醉的鱼，这是玻璃体内注入与吗啉，轻轻包裹在湿纸巾（左侧面板）。纸巾应包括鳃，但并不妨碍眼（中间面板）。轻轻按下正极腹侧部的眼睛，让眼睛的背半旋转插座。从眼睛的背侧半负极约1毫米，并开始电击（右图）。 <img alt="图4。" src="/files/ftp_upload/3603/3603fig4.jpg" /> 图4比较而玻璃体内注射一个啉里沙明标签（一），和视网膜与里沙明标签的吗啉（二）玻璃体内注射和电穿孔电穿孔的一个视网膜的共聚焦图像。里沙明标签发现，所有的视网膜层和细胞类型。活性氧，棒外段;外核，外核层的INL，内核层;保利协鑫，神经节细胞层。 <img alt="图5。" src="/files/ftp_upload/3603/3603fig5.jpg" /> 图5比较PCNA的免疫定位与控制啉（左侧面板），并与抗PCNA啉（右图）前的光诱导感光细胞死亡的电穿孔视网膜电穿孔视网膜共焦图像。暗适应成年白化斑马鱼被放置1，2，或3天的吗啉电击后在不断强烈的光线。控制啉未能压制在光损伤视网膜PCNA表达增加。抗PCNA啉有效撞倒通过不断强光损害三天PCNA蛋白表达。 <img alt="图6。" src="/files/ftp_upload/3603/3603fig6.jpg" /> 图6。示意图（左图）为P眼眼球摘除rocessing和cryosectioning，这是沿背腹轴。粉红色的阴影框（右图）显示与使用这种技术的蛋白质击倒的最大金额的视网膜区域。蓝色的阴影框代表，是最常见的电击事件破坏的地区。眼睛的方向标记（一）前，后（P），（四）背，腹（五）。

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Sci U.S.A. 101, 16-22 (2004).</li><li>Kustermann, S., Schmid, S., Biehlmaier, O., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+excitability,+and+transfection+of+retinal+neurons+in+an+organotypic+culture+of+mature+zebrafish+retina.">Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina.</a> Cell Tissue Res. 332, 195-209 (2008).</li></ol>

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最近，两个芯片分析研究基因表达的变化发生在光受损的视网膜，或手术切除视网膜修补10，16再生。这两项研究显示，展出众多的基因表达显著变化，增加或减少，这可能是针对不同的事件发生在视网膜再生的，如米勒胶质细胞重新进入细胞周期进入，要求，继续增殖和迁移神经祖细胞受损的视网膜层，并到正确的神经细胞类型的神经祖细胞的分化。虽然这两个芯片的数据集的直接比较，揭示候选人在这些细胞的活动可能涉及的基因，这些基因及其编码的蛋白质最终必须进行测试，以确定它们是否是必要的神经再生。在这里，我们描述了一个强大的丧失功能的方法来conditio成年斑马鱼视网膜的利益应受击倒的蛋白质。这项技术已用于研究外和细胞内的蛋白质，以及信号分子，转录因子和DNA 复制 17-19所需的蛋白质。因此，这种方法有很大的帮助我们基本在斑马鱼成人视网膜再生的分子机制的理解。 我们测试了多个电参数（电压，脉冲数，脉冲之间的时间）前取得一个成功的协议。一个以前的报告中使用的电穿孔进入再生斑马鱼尾鳍morpholinos在低电压（15 V） 的 20 10个脉冲。因为成年斑马鱼视网膜是由多个细胞层包围，不容易接触到镊子电极，在鱼翅中所使用的参数是不成功的，在有效地进入成人视网膜电穿孔morpholinos。相反，高电压（100伏）经常水库ulted死亡的鱼。近日，5脉冲80 V的描述electroporate 21小狗新生小鼠视网膜质粒DNA。我们发现，略少激烈2脉冲75 V的morpholinos成功电击到100％经处理后的鱼的生存与成年斑马鱼视网膜。此外，我们发现，去除最外面的（或最外部的）角膜组件，大大提高了电穿孔效率。增加脉冲数，可以消除消除这个组织的需要，但也可能导致更大的组织损伤。广泛的控制的研究表明，这些参数没有造成任何显著的视网膜细胞死亡或改变在感光再生 19中观察到的特定的细胞反应。增益功能的研究是目前无法使用这种技术在体内 ，由于我们无法一贯electroporate所有R大型核糖核酸etinal细胞。然而， 在植体内和斑马鱼视网膜的小鼠视网膜质粒电穿孔进入的成功表明，它仍然是一个在斑马鱼 21， 22的可能性。 这项技术的优势之一是电击出现，有效地引入不同的视网膜细胞类型的吗啉。然而，一个潜在的弱点，电击有效地传递到只有背和视网膜中央区域啉。朝着良好的目标是在所有图层的视网膜结果腹半的第二次电击事件，但往往损害结果。这种空间的限制，可能是由于电极的形状和位置。定制设计的杯形电极，可放在眼部周围的使用可能会改善这一弱点。这啉交付空间的限制使用这种技术，并排除使用检测SUCERG的分析h的，就需要一个全球评估的视网膜。应该指出的是，与任何啉实验，最好是确认你的结果，使用第二个，非重叠啉靶mRNA。在某些情况下，很可能也electroporate两个不同的morpholinos同时击倒了两个不同的蛋白质表达。我们证明撞倒的Pax6a和Pax6b蛋白质的表达，单独和组合 18这一点。 虽然我们展示了使用这种技术，我们的光损伤模型，它可能可以用来研究其他损伤模型2-8再生或用于研究蛋白质的功能完好的视网膜。例如，morpholinos电击可以用来击倒的具体通道或在神经节或无长突细胞的信号转导分子的表达，然后研究这些信号的功能在视觉处理的途径。然而，作为morpholinos只影响新的蛋白质的翻译，必须要等到发生内源性蛋白质周转才可以执行一个试验。根据蛋白的稳定性，范围可以从几个小时到天18。

<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 </td><td>保得国际</td><td> CUY21EDIT或CUY21SC </td><td>这两个单位的工作本议定书</td></tr><tr><td>直径3毫米桨电极</td><td>保得国际</td><td> CUY 650 - P3 </td><td></td></tr><tr><td>吗啉</td><td> GeneTools，有限责任公司</td><td></td><td>吗啉应当自定义设计，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;稀释1:1000鱼制水过程中，安乐死1:500 </td></tr><tr><td>杜蒙＃5镊子</td><td>世界精密仪器</td><td>14095 </td><td>任何工作＃5镊子</td></tr><tr><td>杜蒙＃5B镊子角度提示</td><td>世界精密仪器</td><td> 500234 </td><td>用于enucleate眼睛以下安乐死</td></tr><tr><td>蓝宝石刀片，双边枪，1毫米宽的蓝宝石刀柄， </td><td>世界精密仪器</td><td>刀片:500314 手柄:500317 </td><td></td></tr><tr><td>汉密尔顿注射器33计钝针可拆卸</td><td>汉密尔顿公司</td><td>注射器:87930 针:7762-06 </td><td>删除，用注射器针头和替换33号钝针</td></tr></tbody></table>

in vivo electroporation of morpholinos into the adult zebrafish retina

许多破坏性的遗传性眼疾造成逐步和不可逆转的失明，因为人类无法再生死亡或患病的视网膜神经细胞。相比之下，成年斑马鱼视网膜拥有强大的能力，自发地再生是在各种不同的视网膜损伤模型，包括视网膜穿刺术，化学消融，集中的高温，和强光治疗1-8失去任何神经类。我们的实验室广泛的特点不断强光处理后玻璃体内注射哇巴因2，5，9的内层视网膜神经元再生光感受器。在所有情况下，居民米勒的神经胶质细胞重新进入细胞周期的产生，这将继续增殖，迁移到适当的视网膜层，在那里他们不足的神经元分化的神经祖细胞。 我们的特点在五个不同阶段的光，DAMA再生GED视网膜特定的细胞反应突出。我们确定了在每个阶段的视网膜再生多个差异表达的基因，通过基因芯片分析 10 。这些基因中的许多人还眼发展的关键。为了测试每个候选基因/蛋白的作用，在视网膜再生的，我们需要制定一个有条件限制，只能在次候选人蛋白的表达，在成人视网膜再生的方法。 啉寡核苷酸被广泛用于研究特定蛋白质的功能丧失，在斑马鱼，爪蟾，鸡，鼠的发展，和人类异种移植肿瘤11-14。这些修改后的寡核苷酸碱基互补的RNA序列，以阻止靶RNA的剪接或翻译。 Morpholinos稳定在细胞中，可以消除或"击倒"为三至五年天12蛋白的表达。 在这里，我们描述了一个有效击倒目标在成年斑马鱼视网膜蛋白的表达方法。这种方法采用里沙明标记的反义，到成年斑马鱼眼玻璃体注射morpholinos。吗啉是利用电极钳，然后到背侧和中央视网膜的所有类型的细胞电穿孔。里沙明提供啉电击负责，并可以可视化评估的视网膜细胞啉的存在。 有条件击倒在视网膜上可以用来研究在再生过程中的不同时间的特定蛋白质的作用。此外，这种方法可用于研究二阶神经元的视觉传导和视觉处理等过程中的特定蛋白质，在未损坏的视网膜中的作用。

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

有条件击倒在成年斑马鱼视网膜的目标蛋白质的表达，是一个方法，它涉及到玻璃体内注射反义morpholinos和电穿孔进入视网膜。由此产生的蛋白质是撞倒了好几天​​，这使得检测蛋白质的作用，在再生或完整的视网膜。

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-adult-zebrafish-retina

Research

JoVE Journal

Biology

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

19.1K 次观看.  Wayne State University School of Medicine. 有条件击倒在成年斑马鱼视网膜的目标蛋白质的表达，是一个方法，它涉及到玻璃体内注射反义morpholinos和电穿孔进入视网膜。由此产生的蛋白质是撞倒了好几天​​，这使得检测蛋白质的作用，在再生或完整的视网膜。

视频: In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

器官型培养的成年兔视网膜

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

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 ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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 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 ...