原材料要感谢他们为帮助制定战略，已逐步形成夹层技术在成年斑马鱼在过去的十年的无数贡献Zon和戴维森研究实验室的成员。此外，我们希望表达我们的感激之情圣母院斑马鱼研究中心提供优秀正在进行的畜牧业照顾我们的斑马鱼殖民地的工作人员。 RAW，得到了美国国立卫生研究院NIDDK授予奖DK083512的科研经费，并从圣母大学的慷慨实验室的启动资金。

1。成年斑马鱼肾脏的解剖，从一个不固定的动物样本<ol><li>选择解剖年龄在3-6个月之间的成年斑马鱼。 </li><li>安乐死的鱼放入一盘0.2％Tricaine pH值7.0，约4-5分钟，仔细观察看到的鳃停止移动和心脏停止跳动。 </li><li>用勺子，电梯从Tricaine洗澡的鱼在少量溶液，倾倒的解决方案，轻轻地放在纸巾上的动物。 </li><li>使用一双解剖剪刀锋利，以消除动物的头，作出削减仅次于鳃鳃盖（图1，路径A）。丢弃在生物危险废物的容器的头部。 </li><li>用解剖剪刀，长腹切口，从头部开始，终止和尾鳍的基础（图1，路径B）尾巴，动物的身体打开。 </li><li>用一双解剖镊子/钳取出的动物内脏器官，和生物危险废物。小心勿刷背体壁，因为这是肾脏的位置（图2）。如果你选择了一个女性的鱼，你将需要舀体腔，可最容易使用产钳或镊子的角度对发展鸡蛋。 </li><li>使用夹层针针身体墙壁打开一个夹层托盘和角度可以可视化的针脚，使肾脏。肾脏将拥有一个形状特征（头部，躯干/鞍，尾）和色彩，穿插黑色色调的色素沉着（图2）。您还可以看到充满外周血，跑下来的器官结构的中线主动脉。 </li><li>用细镊子从背侧体壁分离肾脏，使用一对镊子，解除肾为你单独由结缔组织构成的器官。 </li><li>进一步的活体细胞的研究，如流式细胞仪准备含有1X PBS离心管，轻轻放入所需的车辆肾脏。 </li></ol> 2。成年斑马鱼肾脏从一个固定的动物样本的制备及夹层<ol><li>准备了4％paraformaldehyde/1X PBST的解决方案。熬的解决方案，以溶解到溶液中的煤灰，然后冷却到4 ° C的混合物，并添加0.1％二甲。我们曾从使用新鲜的煤灰解决方案，而不是冻结等分组织保存的最成功的。淹没在夹层的成年斑马鱼做出足够的解决方案。 </li><li>填充足够的固定解决方案淹没在成年斑马鱼解剖一个夹层托盘。我们执行的后续步骤，在显微镜下定位托盘，可放置在化学罩，以尽量减少暴露在固定的气味。 </li><li>选择解剖年龄在3-6个月之间的成年斑马鱼。 </li><li>安乐死0.2％Tricaine pH值7.0约5分钟，成菜的鱼，仔细观察，看到的鳃和心脏停止跳动。 </li><li>用勺子，电梯从Tricaine洗澡的鱼在少量溶液，倾倒的解决方案，轻轻地放在纸巾上的动物。 </li><li>使用一双解剖剪刀锋利，以消除动物的头，作出削减仅次于鳃鳃盖（图1，路径A）。丢弃在生物危险废物的容器的头，并立即放入动物的煤灰含有夹层托盘。 </li><li>使用解剖剪刀打开动物的身体，长腹切口，开始从头部和尾部尾鳍的基础（图1，路径B）终止，并保持身体部分淹没在的过程。 </li><li>用一双解剖镊子/钳取出的动物内脏器官，和生物危险废物。小心勿刷背体壁，因为这是肾脏的位置（图2）。 </li><li>使用夹层针针打开身体的墙壁，和角度可以可视化的针脚，使肾脏。肾脏将拥有一个形状特征（头，鞍，尾）和色彩，穿插黑色色调的色素沉着（图2）。您还可以看到充满外周血，跑下来的器官结构的中线主动脉。 </li><li>一夜之间修复的样品，托盘放置在4 ° C当您安排所需的样品做。 </li><li>第二天，从托盘中取出的煤灰解决方案，并取代1X PBST。 </li><li>用细镊子仔细分离肾脏背侧体壁，使用一对镊子，解除肾为你单独器官的结缔组织构成。肾组织柔软，柔韧，从固定液DMSO，使它能够在一块的整个肾脏器官解剖。 </li><li>使用宽孔转移吸管放入离心管的肾。 </li><li>肾脏，现在可以进一步处理， 进行原位基因表达研究的基础上所需的研究的组织学或整装。 </li></ol> 3。代表性的成果： 图解图1中的步骤表明解剖过程。肾脏可根据其形状特征和着色，以及对动物的体腔的背墙解剖位置确定，如图2所示。解剖一个固定的肾脏样品，整个装入原位杂交后，可用于本地化的一个感兴趣的基因的表达，如在图3所示。 <img src="/files/ftp_upload/2839/2839fig1.jpg" alt="图1"/> 图1：程序，使解剖斑马鱼成人肾脏的直接访问 。 （一）安乐死的斑马鱼是解剖一个循序渐进的方式（用数字和箭头表示）给研究者完整的肾脏器官的最简单的访问。腹腔的图片前，（B）和（三）去除腹部器官。 <img src="/files/ftp_upload/2839/2839fig2.jpg" alt="图2"/> 图2：在一个不固定的样本的斑马鱼肾可视以下拆除在腔体内的器官，肾脏出现一个单一的，扁平的器官是通过结缔组织 （一）背体壁贴壁，并已系统化（二）以显示其解剖形状。 <img src="/files/ftp_upload/2839/2839fig3.jpg" alt="图3"/> 图3： 在执行对整个成人肾脏器官的原位杂交装载 。 （一）cadherin17成绩单被发现在成人肾肾肾小管，扩大（一）（B）mafba成绩单本地化近端肾细胞类型，在成人的肾（比较面板整个肾小管一），（ B“ ）mafba表达的放大视图显示成绩单专门的足细胞（P）的细胞的血液过滤器（肾小球）和近曲小管（PCT）的本地化。

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没有利益冲突的声明。

成人干细胞都充满活力的重要组成部分，保持成年体形式 1 。成人干细胞也能起到抵制破坏，在疾病状态下机体所产生和成人干细胞的功能障碍或丧失，可导致器官功能衰退，并已牵连到驱动器的恶性肿瘤，并促进老化。成人干细胞表现出细胞分化的细胞类型的属性，区分他们。细胞分裂后，成人干细胞能够自我更新和祖细胞的生产，这反过来可以带来明显的区别的后代。有极大的兴趣，了解的途径，调节细胞命运的决定和成人干细胞的效力，因为其重要作用的组织动态平衡 1 。例如，许多科学家目前正在设法确定的信号，保持各种成体干细胞在自己的优势种群，或专门的微环境，以及如何这个利基是通过体液因素的影响。 成人的造血干细胞（HSCs）是多能干细胞，燃油的不断复兴的血细胞在动物 8 。造血是一个充满活力的过程，就是在脊椎动物的有机体的生命不断持续，因为成熟分化血细胞类型是短命的，因此必须定期补充。由于它们的分化，造血干细胞自我更新并产生了一系列的祖细胞，引起红系，巨核细胞，淋巴细胞和髓细胞谱系提供这个关键的需求。这些类型的血细胞中的每一个执行了独特的功能，在流通领域，包括组织提供的气体交换，密封血管的血凝块形成的伤害，或对入侵的病原体防御机制的一种方式，。虽然已多年公认的造血干细胞，过多的迷人问题仍然未对这些惊人的细胞。最近的研究确定了不同的长期自我更新的属性，并澄清利基元件和信号调节HSC 的行为8的造血干细胞的子类。斑马鱼是目前造血功能的研究范式的基石，根据对这种动物模型8的遗传和分子属性。利用斑马鱼的研究，导致有关HSC的生物学是保守的小说，像哺乳动物的高等脊椎动物中， 强调鱼血调查8的生物医学相关的见解。 从历史上看，有关造血干细胞知识的探险发现，如果身体其他器官是由成人干细胞维持铺平了道路。现在众多的成人干细胞群赞赏存在，在不同的结构，从大脑到肠，皮肤，骨骼肌。在干细胞领域继续努力，寻求以确定如何成人干细胞和分化的细胞，甚至其他类型，可以显示各种设置，增强药性。 对于肾脏有肾干细胞是否存在16肾科之间的大辩论。独立研究结果已经证明在哺乳动物中，具有不同程度的再生潜力的肾细胞的数量，但这些报告的凝聚力理解尚未实现。有趣的是，有强有力的证据来支持这一概念，很多鱼类物种具有细胞可以再生受损的肾强劲燃料，以及在成年期9成长的全新肾。最近的工作已经确定了在斑马鱼10,11成人肾脏干细胞的分子标志，并展示了这些所谓的肾干/祖细胞（RPC） 的 11自我更新的药效。未来的研究需要，以确定是否类似肾细胞在哺乳动物中存在。然而，有关的细胞和分子机制，调节鱼的RPC继续调查可能会提供如何可能在哺乳动物肾脏刺激再生功勋的洞察力。很明显，仍然有许多关于RPC的理解和实施的许多工具，现在提供的斑马鱼研究员可以解决这些有趣的问题。 在这个协议中，我们描述了我们的方法为肾成年斑马鱼的解剖鉴定和清扫。其他的步骤可以用来解剖肾，如使用夹层剪刀与腹侧切口打开腹腔，然而，我们所遇到的最成功的访问时腹壁固定开放和头部被删除整个肾脏。血，肾的研究人员都希望隔离，并与这些类型的细胞工作可以使用清扫方法。应当指出，我们所描述的方法，隔离和workin与斑马鱼的成人肾脏G是肯定不限于那些希望追求成人干细胞生物学的问题，作为本机关的夹层可以方便那些追求从生理老化的议题进行调查的研究人员研究的科学家。此外，这种方法可以加上其他如转基因技术，所以作为分子标签，然后净化和研究离散的从野生型或基因突变斑马鱼11的血液或肾细胞亚群。展望未来，干细胞和祖细胞类型等详细的净化程序是至关重要的，以获得有关如何调节自己的行为是知识，操作测试，如移植或祖细胞培养的基础构成，这些细胞的潜能和身份的定义。在造血祖细胞培养方法的最新进展为研究打开许多新的途径，可适应培养17例肾人群。斑马鱼的化学遗传学已成功途径，调节造血干细胞，并在各种情况下的肾祖 18-20，将继续成为一个有用的途径与上述细胞生物学技术结合测试鉴定。两者合计，有一个使用斑马鱼模型的血液和肾脏病领域的开创性贡献的数量，并继续在这些领域的研究承诺挥动进一步变革的见解，在未来几年。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>解剖剪刀</td><td> Roboz </td><td> RS - 5882 </td></tr><tr><td>解剖钳</td><td> Roboz </td><td> RS - 5010 </td></tr><tr><td>夹层直角钳</td><td> Roboz </td><td> RS - 5058 </td></tr><tr><td> Tricaine </td><td>西格玛</td><td> E10521 </td></tr><tr><td>多聚甲醛</td><td>电子显微镜</td><td> 19210 </td></tr></tbody></table>

dissection of the adult zebrafish kidney

在成人干细胞生物学的新兴领域工作的研究人员设法了解规范的行为和干细胞的功能，在正常的动态平衡和疾病状态的信号，。成人干细胞的认识，具有广泛的深远影响， 为再生医学的1未来。例如，更好地了解成人干细胞生物学的知识，可以方便的触发机关是医治自己， 甚至是建立在体外的器官可以移植到人类1方法治疗策略的设计。斑马鱼已成为一个强大的动物模型，为研究脊椎动物的细胞生物学2。已经有广泛的文档和胚胎发育中的斑马鱼3的分析。直到最近，科学家试图文件成人解剖和手术剥离技术4，因为一直在斑马鱼的社会进步运动，以扩大本研究的有机体到成人的研究中的应用。例如，有扩大使用斑马鱼进行调查的成人干细胞群生物学和复杂的疾病，如癌症5成人模式的利益。从历史上看，隔离的斑马鱼成人肾脏已经为研究造血， 肾脏是6,7鱼血细胞生产的解剖位置。肾脏是由肾功能arbo​​rized安排的单位，整个空间是分散的造血组织，包围。造血组成部分造血干细胞（HSCs）和他们的后代居住在肾脏，直到他们临终区分8。此外，它现在是赞赏，肾干/祖细胞组（RPC）的也居住在斑马鱼的肾脏器官，使肾脏的再生和增长，为其他鱼种 9-11观察。鉴于这一新发现，斑马鱼的肾脏是一个器官，房子的成人干细胞生物学研究的两个令人兴奋的机会的位置。它是明确的，许多悬而未决的问题，可以担任这个实验系统。为了鼓励这一领域的扩展，它是有益的文件详细的可视化方法，然后隔离成年斑马鱼的肾脏器官。该协议的细节，不固定和固定动物的成年肾解剖的过程。可用于解剖的肾脏器官，造血功能和肾功能的干细胞和它们的后代使用，如组织学，荧光激活细胞分选（FACS）11,12，13,14表达谱，并移植11,15成立技术分离和鉴定。我们希望传播，这个协议将提供的知识，以实现更广泛的使用，最终可以翻译为人类应用的斑马鱼研究的研究人员。

Watch this Scientific Journal Video about Dissection of the Adult Zebrafish Kidney at JoVE.com

Dissection of the Adult Zebrafish Kidney

斑马鱼的肾脏是成人肾和造血干/祖细胞，并代表一个绝佳的机会，这些细胞类型和他们的后代在脊椎动物模式生物研究。在这里，我们展示了详细的解剖程序，使研究人员识别和手术去除成年斑马鱼的肾，可用于细胞分离，移植，肾和/或血液细胞群表达的研究，如应用。

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 ...

Research

JoVE Journal

Biology

36.5K 次观看.  University of Notre Dame. 斑马鱼的肾脏是成人肾和造血干/祖细胞，并代表一个绝佳的机会，这些细胞类型和他们的后代在脊椎动物模式生物研究。在这里，我们展示了详细的解剖程序，使研究人员识别和手术去除成年斑马鱼的肾，可用于细胞分离，移植，肾和/或血液细胞群表达的研究，如应用。

视频: Dissection of the Adult Zebrafish Kidney

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

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

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, ...

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 ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...

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 ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...