我们感谢欧共体与缝合帮助廖和R. Ethier和L. GYR斑马鱼的护理。 AJD是由哈佛干细胞研究所，美国肾脏病学会，美国国立卫生研究院/ NIDDK（P50DK074030）的支持。 CQD是由美国马萨诸塞州总医院医学发现基金的支持。

1。空调的非转基因收件人鱼类。 <ol><li>选择在系统中用于移植的最大公鱼。 </li><li>将在0.02％Tricaine的鱼（pH值7），约3分钟，或直至它停止游泳，但没有这么长，心脏停止跳动。 </li><li>麻醉鱼湿纸巾放在一条长凳上方。 </li><li>使用胰岛素注射器针头2​​0 UL水成鱼的腹腔腔注入庆大霉素的40微克。对于肾功能再生的研究，这一步很重要，诱使一个有利的环境，增强植入。 </li><li>鱼放入水系统的恢复。 </li><li>鱼可被驯服的和非常敏感的震动等外部刺激。经过3个小时的恢复，治疗25伽玛射线的灰色的鱼。 </li><li>将没有食物移植前3天的鱼放回系统。 </li></ol> 2。祖供体细胞的制备，用荧光标记（EGFP或者mCherry）基因标记。 <ol><li>手术过程前，准备捐助移植的细胞，并保持他们在冰上。 </li><li>对于肾功能的研究中，我们使用全肾从TG（cdh17 EGFP）的骨髓细胞转基因株系，其中包含肾脏和造血祖细胞。这些祖细胞的基因标记，并仅表达EGFP的，如果他们嫁接和肾小管上皮细胞分化成。也可用于其他类型的标记的祖细胞，如造血干细胞或TG（fli1a：EGFP）angioblast祖细胞移植的TG（SCL：EGFP ）。 </li><li>放置5分钟，他们在0.2％Tricaine安乐死3成年转基因鱼。 </li><li>使用消毒刀片去除头部和尾部，和解剖剪刀，沿腹方一个正中切口。 </li><li>在一个立体，删除所有在腔体内，包括镊子鱼鳔的五脏六腑。不小心取出肾脏，这是一个单位和色素的器官，腔背墙。 </li><li>接下来，从背墙解剖肾脏，放入1.5 mL离心管含300冷磷酸盐缓冲生理盐水辅以2％胎牛血清（1X PBS / 2％FCS）UL。 </li><li>使用杵剪切肾脏，直到它变得同质化。 </li><li>将一个50毫升猎鹰管内40 UM细胞过滤器和转移的细胞过滤器上的电池解决方案，让松散的细胞进行筛选，通过猎鹰管。 </li><li>使用杵轻轻涂抹，留在过滤器上，进一步打破了细胞组织的大块。 </li><li>留在“猎鹰”管细胞溶液，并用1.2毫升的1X PBS / 2％FCS的过滤器，以收集过滤器的残余细胞。 </li><li>转让4 ° C时，300 RCF 5分钟到一个新的1.5 mL试管和离心的电池解决方案。 </li><li>用1.5 mL的1X PBS / 2％FCS中清洗细胞沉淀，并通过一个新的过滤器，过滤掉粘细胞的团块，离心再次。 </li><li> 200 UL的1X PBS / 2％FCS重悬细胞沉淀，并转移到PCR管。 </li><li>离心机PCR管“（4℃，300 RCF，1分钟），除去上清液。 </li><li>重悬细胞中的残留上清获得2​​-5 UL最终体积和存储在冰上。 </li><li>最后蜂窝产量约200万个肾细胞。 </li><li>计数与血球细胞的数量，并调整浓度至5 × 10 5细胞每微升。 </li></ol> 3。汉密尔顿注射器装入电池解决方案。 <ol><li>汉密尔顿注射器无菌水冲洗3次。 </li><li>注射器冲洗干净，用75％乙醇3倍。 </li><li>注射器冲洗用1X PBS / 2％FCS的3倍。 </li><li> 1 UL细胞溶液中含有约5 × 10 5细胞注射前，然后装入注射器。 </li></ol> 4。直接入肾移植的祖细胞。 <ol><li>在0.02％Tricaine的鱼约3分钟，或直至停止移动，但不会太长，心脏停止跳动的麻醉空调收件人鱼。 </li><li>立体显微镜下，奠定了纸巾上，头向左侧，腹侧的鱼，以干燥移植方面。 </li><li>然后滚鱼揭露干边。 </li><li>使用消毒镊子，取出立即去鳃后在该地区的尺度。 </li><li>用手术刀在这方面的一个1厘米中线外侧切口，同时稳定的组织用镊子。 </li><li>使用镊子，公开的组织，同时继续深度不够，使切口揭露头肾。 </li><li>如果有大量出血，防止肾脏的可视化，euthenize鱼，并开始与其他收件人鱼。 </li><li>虽然撬组织公开揭露头肾，头肾直接注入1单元解决方案UL。 </li><li>然后，在同一时间正在检索注射器针头，松开镊子，使组织分为放回原处，以防止泄漏注射的细胞。 </li><li>握住缝合针一针驱动程序和皮尔斯通过切口两侧的肌肉。 </li><li>铁2节，并切断多余的缝合。只有一个单一的缝合需要一个1厘米的切口。 </li><li>包含5个小时的百万分之10的Tricaine提供一个平静的镇痛效果的鱼放置在鱼水之鱼。 </li><li>将系统恢复正常喂食鱼背。 </li><li>经过7-18天的恢复，我们解剖鱼和生活EGFP荧光分析肾祖细胞植入。 </li></ol> 5。代表性的成果： 移植的总体战略是在图1所示。对于我们的肾脏研究，肾脏的解剖和EGFP荧光分析。被检测为早7天移植后的肾祖细胞植入和分化。由多个集群的EGFP +肾上皮细胞（图2）存在，这是显而易见的的。移植后18天，我们发现，平均24 EGFP +肾，这表明，供体细胞已经形成了新的肾组织（图3 ）。为了证明长期植入，我们解剖一个收件人鱼59天移植后（图4）。这种鱼有许多肾单位，在注射部位（红色箭头），除了在遥远的地方（白色箭头）的多个肾，表明肾祖细胞迁徙。这种技术是更快和更有效相比，当细胞注射进入血液循环通过心脏，它需要大约3个月检测植入。 <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="图1"/> 图1。辐照抑制细胞移植的免疫排斥反应的移植。非转基因的收件人鱼的整体计划。三天后，切口是鱼和捐助者的祖细胞的侧翼暴露的头肾注入。关闭切口缝合和鱼放入回收系统。收件人鱼肾脏在稍后的时间点解剖和分析，植入和祖活动。 <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="图2"/> 图2。祖细胞植入后七天 。肾祖细胞植入头肾在移植后7天注射方检测，集群的EGFP +肾小管上皮细胞的存在（插图-放大查看）确定。 <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="图3"/> 图3。祖细胞植入后18天 ，移植后18天之后，我们发现24 EGFP的平均捐助者派生肾（插入-放大个人EGFP +肾） 。 <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="图4"/> 图4。植入后59天祖细胞的迁移 。我们继续检测祖细胞移植后长期植入，即使经过59天的（红色箭头头） 。在稍后的时间点，捐助派生肾站点检测到远离注射液（白色箭头头）的网站，与祖细胞的迁移一致。

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

细胞移植技术是再生研究的关键，是黄金标准测量干细胞活性测定。在这里，我们报告，直接进入成年斑马鱼肾移植的细胞，以确定干细胞和祖细胞的活性的方法。此过程涉及通过一个切口，肌肉，头肾，其次是直接注射入肾细胞暴露。封闭，单一的缝合切口和经营鱼有90-100％的存活率。在我们的实验，检测嫁接肾祖细胞移植的鱼在80-100％，移植后7天。这种技术的一个限制是，切口附近的背主动脉，可以很容易地通过手术刀破裂。然而，一旦掌握，可减少出血发生率小于1％。适应我们的方法可以提供其他类型的细胞（血，间质或血管）以及小分子对肾脏。这项技术的发展将有助于推动再生研究，并允许干细胞和祖细胞的潜力，在体内进行评估。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>胰岛素注射器针头（28计） </td><td>碧迪</td><td> 329461 </td></tr><tr><td>细胞过滤器（40 UM） </td><td>碧迪</td><td> 352340 </td></tr><tr><td> Tricaine </td><td>西格玛</td><td> A5040 </td></tr><tr><td>汉密尔顿注射器（26压力表） </td><td>西格玛</td><td> 20734 </td></tr><tr><td> Ethilon缝合（尺寸9-0） </td><td>爱惜康</td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

再生医学的基础上，干细胞或祖细胞移植到受损的组织， 具有潜在的治疗1慢性疾病的范围广泛。然而，大多数器官是不容易接触，必然需要制定手术的方法来获得这些结构。在这个视频文章中，我们描述了，直接进入成年斑马鱼，一个流行的模型来研究再生和疾病2肾移植细胞的方法。收件人鱼是预先条件辐照抑制免疫排斥反应注射的细胞3。我们演示了如何在头肾，可以通过直接在器官的细胞注射，在鱼的侧面的外侧切口暴露。使用荧光标记的全肾骨髓细胞，其中包括一个肾功能和造血前体的混合人口，我们表明，肾祖细胞可以嫁接，并分化成新的肾组织 - 任何基于细胞再生治疗的金标准。适应这种技术可以提供肾脏以及其他内脏器官和纯化干细胞或祖细胞和/或小分子，进一步增强了作为一个通用的模型来研究再生医学的斑马鱼。

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

细胞移植是一个研究组织的再生和发展疾病的细胞疗法的基本技术。在这里我们展示一个显微外科技术，允许到成年斑马鱼的肾脏的基因标记的细胞直接移植。

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 ...

transplantation-of-cells-directly-into-the-kidney-of-adult-zebrafish

Research

JoVE Journal

Biology

14.9K 次观看.  Massachusetts General Hospital. 细胞移植是一个研究组织的再生和发展疾病的细胞疗法的基本技术。在这里我们展示一个显微外科技术，允许到成年斑马鱼的肾脏的基因标记的细胞直接移植。

视频: Transplantation of Cells Directly into the Kidney of Adult Zebrafish

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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

补肾胶囊对糖尿病小鼠的胰岛移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

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 ...

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 ...