这项工作是支持由Alex的柠檬水摊子基金会（DML），美国癌症协会（DML），麻省总医院霍华德·古德曼奖学金（DML），和美国国立卫生研究院授予R24OD016761和1R01CA154923（DML）。 CNY流式细胞仪的核心和流图像分析，共享仪器的授权号1S10RR023440-01A1。 IMT是由葡萄牙科学基金会和科技（ - FCTFundação第一个西恩西亚éTECNOLOGIA）的奖学金资助。 QT是由中国国家留学基金委资助。我们感谢安吉拉Volorio对她有益的意见和建议。

所有动物的程序批准了美国马萨诸塞州总医院的小组委员会在研究动物护理，根据协议＃2011N000127。 第1节骨骼肌细胞移植到成人RAG2 E450fs纯合突变斑马鱼 1.准备的成年斑马鱼供体骨骼肌细胞<ol><li>获得转基因成年斑马鱼已荧光标记的肌肉。在这个实验中，30α 肌动蛋白，RFP捐赠鱼25被用来移植每个收件人鱼1×10 6个细胞。 </li><li>在1.6毫克牺牲捐助斑马鱼/毫升三卡因磺酸（MS222），10分钟或直到没有动静厣是显而易见的。 </li><li>将捐助鱼吸水纸巾和消费用干净的刀片背部的肌肉。切应靠近肛门以45°角最大化的组织集合（如在图1A中指出的</strong&gt;）。放置解剖组织到一个干净的10cm培养皿。 </li><li>加入500微升的悬浮缓冲液（预冷却的0.9倍磷酸盐缓冲盐水（PBS），补充了5％胎牛血清（FBS））向解剖组织。最多10个捐助国斑马鱼可以放在一起本卷。 </li><li>讳言用刀片将组织&gt; 20次，直到细胞在均匀的悬浮液。整个背部肌肉均匀化，包括皮肤，骨骼和鳍。加入2毫升悬浮缓冲液。使用5毫升吸管，磨碎的细胞悬液≥20倍以解离细胞。 </li><li>过滤通过40微米的筛网过滤器的细胞悬浮到50ml锥形管置于冰上。 </li><li>用额外的2.5毫升悬浮缓冲液洗培养皿以收集剩余的组织，并通过同样的过滤器和锥形管过滤，以5毫升（10施主鱼可以每分离物被使用）的最终体积。 注意：皮肤，骨骼和鳍将过滤后排除。 </li&gt; <li>如果适用的话，结合相似悬浮到相同的锥形管中。 </li><li>算使用台盼蓝染料与血球存活细胞的总数。 </li><li>保留500μl的流式细胞术，如果需要的话（可选的，步骤2）。 </li><li>离心细胞悬浮液在1000×g离心，10分钟，在4℃下。 </li><li>弃上清，悬浮细胞在3.33×10 5个细胞/μL（0.9X PBS + 5％FBS）。总共，3微升将每个收件人鱼注射总共每个收件人1×10 6个细胞（第3步）的。 注：小于3微升细胞悬浮液应该被移植到受体鱼。如果细胞数被限制，因为低达5×10 4个细胞每收件人可导致成功植入（ 表1）。 </li></ol>供体骨骼肌细胞的制备2.流式细胞仪分析（选配） <ol><li>从野生型 ，非转基因鱼隔离概述我的肌肉n步1.1。该样品用作阴性对照，是用于设定流式细胞计数门是有用的。 </li><li>添加适当的生存能力染料。例如，添加5微升股票DAPI溶液（500毫微克/微升），以500微升肌肉的准备。涡稍微之前的分析。获得5×10 3〜1×10 4个事件。首先分析野生型对照样品放在门后面的肌肉细胞转基因鱼孤立的分析。 通常在1小时内的肌肉组织解剖后，在这段时间内解剖细胞保留超过60％的存活率（ 图2）进行的流式细胞术分析：注。细胞应保持在冰上在任何时候。使用台盼蓝染料与血球总细胞生存力可以被重新评估移植前。 </li></ol> 3.肌内移植骨骼肌细胞变成成年RAG2纯合突变斑马鱼<ol><li>清洁10μ;升26S G微型注射器通过拉伸并排出10％的漂白剂溶液（5次），然后用70％乙醇（5次），然后接着悬浮缓冲液（0.9X PBS + 5％FBS，10次）。 </li><li>通过加入单滴三卡因甲磺酸盐（MS222，4毫克/毫升储液）到含有在体系水中的鱼，直到厣动作缓慢培养皿麻醉2-4月龄纯合RAG2突变鱼或野生型接受者鱼（作为对照）和鱼类都还在。 注：三卡因麻醉剂量将取决于接受者的年龄斑马鱼和大小。 </li><li>将麻醉收件人斑马鱼的湿纸巾或海绵，用左手面朝上。 </li><li>将注射器针头插入latero-背侧肌肉（参考图1A）。确保注射以45°角进行的。注射3微升每鱼的细胞悬浮液（在步骤1.12中制备）的总计每个收件人1×10 6个细胞。</li><li>小心用塑料勺子恢复注入斑马鱼转移到一个干净的坦克。 </li><li>通过在亮场和荧光显微镜成像麻醉鱼评估收件人斑马鱼为植入率在10，20，30天后移植。 </li></ol>第二节胚胎性横纹肌肉瘤（ERMS）移植到成人纯合子突变RAG2斑马鱼斑马鱼胚胎的DNA 4.显微注射<ol><li>通过消化10微克的DNA用XhoI的，在37℃下进行6小时或O / N线性化RAG2-KRASG12D质粒7。 </li><li>纯化的DNA通过标准的苯酚：氯仿萃取和用乙醇沉淀。重悬在20μl去离子水（替代地，商业的DNA片段纯化柱可以使用）。 </li><li>运行在1％琼脂糖凝胶未消化和消化的DNA，并确定DNA的b的浓度Ÿ光谱仪的读数。 1，1：：另外，在1运行样品5和1:10的稀释液在1％琼脂糖凝胶和量化相比的DNA梯。 </li><li>制备的注射混合在15毫微克/微升消化的RAG2-KRASG12D的DNA在0.1M KCl和0.5倍的Tris-EDTA的最终浓度。在2 NL注射体积中喷射出的最终的DNA量将是30页。 注：最多三个不同的DNA构建体可以有效地共注射在最高60皮克每胚胎的DNA。这些转基因被整合到基因组中，并共表达内显影肿瘤26。 </li><li>注入线性RAG2-KRASG12D成单细胞阶段的胚胎基本上如27到感兴趣的斑马鱼品系（ 图1B）。注射应在细胞而不是为了更高的效率来执行在蛋黄。在该实验中，双转基因AB-应变; Myf5的-GFP，mylpfa-mCherry使用。使用标准的饲养P提高斑马鱼rotocols 28。 注：注塑生存通常取决于所用的斑马鱼品系。平均来说，注射的胚胎的30％将发展ERMS。 300-600胚胎应每个实验，以确保用于移植和分析所产生的足够的GFP阳性和mCherry阳性原发性肿瘤注射。 </li></ol> 5.筛选斑马鱼幼虫主要ERMS <ol><li>观察注入斑马鱼从10至30天注射后对外部可见主ERMS的出现。 </li><li> 30天注射后，通过加入单滴三卡因甲磺酸酯（MS222 4毫克/毫升储液）到含有鱼系统水直到厣动作缓慢和鱼仍培养皿麻醉收件人斑马鱼。 注：三卡因麻醉剂量将取决于接受者的斑马鱼的年龄和大小。原发性肿瘤的斑马鱼需要较低剂量的三卡因的。 </li><li>选择初级ERMS轴承鱼类，Myf5的-GFP阳性和mylpfa-mCherry阳性，用落射荧光显微镜。 </li></ol> 6. ERMS肿瘤准备<ol><li>牺牲选定的主ERMS轴承斑马鱼在1.6毫克/毫升三卡因磺酸（MS222），10分钟或直到没有动静厣是显而易见的。 </li><li>分别处理每个荷瘤斑马鱼。在一个干净的培养皿中放置的鱼和解剖周围用刀片和细镊子（ 如图1B）的肿瘤。传送解剖肿瘤组织到一个干净的培养皿中。 </li><li>加入100微升预冷的0.9倍磷酸盐缓冲盐水（PBS）的补充有5％胎牛血清（FBS）。剁碎的组织用干净的刀片&gt; 20次，直到细胞在均匀的悬浮液。 </li><li>添加900微升的相同缓冲液（0.9X PBS + 5％FBS）中，移液管上下数次以解离应用1000微升过滤移液管尖的细胞。经过40过滤器＃956;米滤网成相应的50ml锥形管中。储存在冰。 </li><li>与一个附加的2-4毫升缓冲液洗培养皿，并经过相同的目滤网并进入相应的锥形管中。 </li><li>离心机在1000×g离心，10分钟，在4℃下。 </li><li>弃上清，重悬在100微升缓冲。 </li><li>算使用台盼蓝染料与血球存活细胞的总数。 </li><li>稀释细胞至所需的浓度，在同样的缓冲液（0.9X PBS + 5％FBS）中。细胞应稀释至5×10 3个细胞/微升移栽每个收件人斑马鱼5微升在总共每个收件人2.5×10 4个细胞。 </li><li>流式细胞计数分析也可以用少量的从步骤6.5的悬浮液，以量化样品中的荧光细胞的相对比例进行。 注：除了100微升细胞悬液（以下步骤3.5过滤）的设置和稀释400微升的0.9倍PBS + 5％FBS停牌流式细胞分析缓冲区。为了确保正确的门，用单一的转基因肿瘤组织或肌肉成年野生型，Myf5的-GFP和mylpfa-mCherry鱼隔离进行进一步的分析。在第1节的第2步进行描述流式细胞本质。 </li></ol> 7. ERMS移植到成人RAG2纯合突变斑马鱼<ol><li>通过绘制在与排出10％的漂白剂溶液（5次），然后用70％乙醇（5次）清洗一个10微升26S G微型注射器，然后接着悬浮缓冲液（0.9X PBS + 5％FBS中的10倍）。 </li><li>通过加入单滴三卡因甲磺酸酯（MS222 4毫克/毫升储液）的入水体系含有鱼直到厣动作缓慢和鱼仍培养皿麻醉收件人纯合RAG2突变鱼。 </li><li>放置麻醉收件人斑马鱼上的湿纸巾或SPONGE，与腹朝上。 </li><li>注入5微升细胞悬浮液进入腹膜腔（每个收件人2.5×10 4个细胞）。 注：注射针应不同肿瘤注射之间如在步骤4.1中所述进行清洁。 5到10微升可以有效地移植腹膜内，这取决于接受者的鱼的大小。肿瘤植入可通过注入1×10 4〜5×10 5个未分类的细胞每收件人鱼（ 表1）来实现。 </li><li>小心地将收件人斑马鱼放入干净的罐用塑料勺。 </li><li>通过在亮场和荧光显微镜成像麻醉鱼评估收件人斑马鱼为植入率在10，20，30天后移植。 </li><li>利用植入鱼下游应用，包括荧光活化细胞分选（FACS）来评估分化状态（ 图3H），标准组织ANALYSIS（ 图3F），成像治疗响应15，和/或串行移植方法包括有限稀释分析11。 </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantable+tumor+lines+generated+in+clonal+zebrafish.">Transplantable tumor lines generated in clonal zebrafish.</a> Cancer research. 66 (6), 3120-3125 (2006).</li><li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291, 293-296 (1981).</li><li>Blackburn, J. S., Liu, S., Langenau, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+frequency+of+tumor-propagating+cells+using+limiting+dilution+cell+transplantation+in+syngeneic+zebrafish.">Quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish.</a> Journal of visualized experiments JoVE. (53), e2790 (2011).</li><li>Tang, Q., Abdelfattah, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cell+transplantation+using+adult+rag2+mutant+zebrafish.">Optimized cell transplantation using adult rag2 mutant zebrafish.</a> Nature methods. 11, 821-824 (2014).</li><li>Zhou, Q., Facciponte, J., Jin, M., Shen, Q., Lin, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+NOD-SCID+IL2rg&#8211;/&#8211;+mice+as+a+preclinical+model+for+cancer+research+and+its+potential+use+for+individualized+cancer+therapies.">Humanized NOD-SCID IL2rg&#8211;/&#8211; mice as a preclinical model for cancer research and its potential use for individualized cancer therapies.</a> Cancer letters. 344 (1), 13-19 (2014).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental biology. 192 (2), 289-299 (1997).</li><li>Langenau, D. M., Keefe, M. D. D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-injection+strategies+to+modify+radiation+sensitivity+and+tumor+initiation+in+transgenic+Zebrafish.">Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish.</a> Oncogene. 27 (30), 4242-4248 (2008).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), 1-5 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Alexander, M. S., Kawahara, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transcriptome+analysis+of+adult+zebrafish+cells+enriched+for+skeletal+muscle+progenitors).">Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors).</a> Muscl, &amp; nerve. 43 (5), 741-750 (2011).</li><li>Motohashi, N., Asakura, Y., Asakura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+transplantation+of+muscle+satellite+cells.">Isolation, culture, and transplantation of muscle satellite cells.</a> Journal of visualized experiments JoVE. (86), 1-7 (2014).</li><li>Gerli, M. F. M., Maffioletti, S. M., Millet, Q., Tedesco, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+induced+pluripotent+stem+cell-derived+mesoangioblast-like+myogenic+progenitors+in+mouse+models+of+muscle+regeneration.">Transplantation of induced pluripotent stem cell-derived mesoangioblast-like myogenic progenitors in mouse models of muscle regeneration.</a> J Vis Exp. (83), e50532 (2014).</li><li>Siegel, A. L., Gurevich, D. B., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myogenic+precursor+cell+that+could+contribute+to+regeneration+in+zebrafish+and+its+similarity+to+the+satellite+cell.">A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.</a> The FEBS journal. 280 (17), 4074-4088 (2013).</li><li>Rowlerson, a., Radaelli, G., Mascarello, F., Veggetti, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+skeletal+muscle+in+two+teleost+fish:+Sparus+aurata+and+Brachydanio+rerio.">Regeneration of skeletal muscle in two teleost fish: Sparus aurata and Brachydanio rerio.</a> Cell Tissue Res. 289 (2), 311-322 (1997).</li><li>White, R. M., Sessa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell stem cell. 2 (2), 183-189 (2008).</li></ol>

The authors have no competing financial interests.

成人背骨骼肌高效且健壮的植入物实现了非常简单的细胞的制备方法，随后通过注射细胞进入的RAG2纯合突变体鱼背肌肉。在一般情况下，肌肉内注射程序是非常强大的，与一些相关死亡的植入程序之后立即，范围从10％至35％，这取决于实验。额外的优化可能会集中在利用较小规格针头注射和使用显微镜和显微注射的固定装置的发展，这将有利于缓解植入细胞。我们的方法也被用来排序的肌肉细胞供体动物，只含有约30％的肌肉祖细胞。使用该标签干细胞和流式细胞仪分离可能会提供丰富的细胞悬浮液导致增加移植到受体的转基因鱼线记者。骨骼肌ÇELLS也可以富集培养移植前，如先前所描述的29。值得注意的是，我们的研究结果还表明，利基建立和捐助肌肉组织分化的步骤发生前10天移植后，建立这种模式的强大和快速的实验平台，以评估肌肉移植和再生。此外，这些实验赤裸裸地与那些在小鼠，其中受伤前的肌肉与心脏毒素或氯化钡需要事先两天植入30,31完成对比。它是可能的移植过程中的过程中产生的损伤针通过刺激生产的受体动物32,33内的再生环境potentiates植入。我们还设想，我们的方法将容易地适应的骨骼肌组织从年轻斑马鱼的移植，允许对影响早期骨骼肌发育的基因突变的评估但导致杀伤力在幼虫阶段。 我们还提供了斑马鱼ERMS中植入了详细的协议，通过腹腔注射到非空调，RAG2纯合突变鱼。这种方法是对膨胀的双转基因的原发肿瘤，而不需要内的同基因转基因品系产生的肿瘤是有用的。我们最近的工作表明，细胞移植的方法提供新颖的实验模型来在体内，其中单个肿瘤可以扩展到数以千计的动物并评估生长，自我更新，和新血管形成的影响15评估ERMS药物敏感性。此外，我们已成功地植入一系列的肿瘤成RAG2纯合突变体的鱼，包括T细胞急性淋巴细胞性白血病，黑色素瘤和ERMS 23。展望未来，我们预计这些线路将是有益的， 在评估癌症重要的功能特性体内包括评估肿瘤内的异质性，侵袭，转移，血管生成和治疗抗性。此外，RAG2纯合突变鱼在光学透明卡斯帕尔株斑马鱼34一代可能会方便不少癌症，这些标志的直接成像。 总共，我们提供的荧光标记的正常和恶性骨骼肌成功植入到成年RAG2纯合突变体的免疫受损的斑马鱼的详细协议。

斑马鱼是用于再生研究的优良模型，因为它们可以再生截肢翅片，以及损坏的脑，视网膜，脊髓，心脏，骨骼肌等组织1。干细胞和再生的研究在成年斑马鱼已主要集中在再生响应于损伤的表征，而识别由细胞移植的各种组织中的干细胞和祖细胞的最近才探讨2。斑马鱼也变得越来越多地用于癌症的研究通过模仿人类疾病3转基因癌症模型的产生- 10。 在癌症的设定，细胞移植方法已被广泛采用并允许重要癌症过程，包括自我更新11，功能异质12,13，新生血管14，扩散的动态评估，治疗反应15，和入侵16,17。然而，植入的细胞通常是由收件人鱼因主机的攻击和杀死移植18免疫防御拒绝。几种方法已被用来克服排斥移植的细胞。例如，受体动物的免疫系统可以瞬时由低剂量的γ射线照射前移植18,19烧蚀。但是，接收者的免疫系统将20天内痊愈辐照后，杀死供体细胞18。可替代地，地塞米松治疗已被用于抑制T和B细胞的功能，提供更长的免疫抑制调节和促进广泛的人类肿瘤细胞植入进行长达30天14。这些实验需要持续用药剂量和有限的实体肿瘤的研究。长期植入实验使用了基因完全相同的同系行20 - 22，在供体和recipi耳鼻喉科细胞是免疫匹配。然而，这些模型需要关注转基因株系进行跨入了同系的背景超过四代产生完全同源的线条。为了避免免疫排斥反应的受体鱼的问题，我们的小组最近开发出一种免疫缺陷RAG2 E450fs纯合突变（ZFIN等位基因指定RAG2 FB101）线已减少T细胞和B细胞功能并允许各种组织23的植入。类似的免疫妥协小鼠模型已被广泛地用于小鼠和人组织24细胞移植。 在这里，我们提出了骨骼肌和胚胎性横纹肌肉瘤（ERMS），儿科肉瘤共享功能与骨骼肌，到新描述RAG2纯合突变的斑马鱼的移植方法。免疫受损成年斑马鱼的可用性扩大了我们进行大规模细胞移植的研究，直接可视化，并在正常和恶性组织评估干细胞自我更新的能力。用这种方法，荧光从成年α肌动蛋白的RFP 25转基因斑马鱼鲁棒嫁接在RAG2标记肌细胞制剂 纯合突变的斑马鱼下注入背肌肉。此外，我们证明移植和扩展的主要Myf5的 - GFP;腹腔注射入RAG2 E450fs纯合突变的斑马鱼mylpfa- mCherry转基因ERMS。这些协议的效用超出所示的例子中，可以很容易地应用到附加斑马鱼再生组织和癌症。

<table border="0" cellpadding="0" cellspacing="0" width="1077">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Tris-EDTA buffer solution 1x</td>
			<td>Sigma-Aldrich</td>
			<td>93283-100ML</td>
			<td>microinjection. Injection mix.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Science Education</td>
			<td>S77375-1</td>
			<td>microinjection. Injection mix.</td>
		</tr>
		<tr>
			<td>XhoI Restriction Enzyme</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>microinjection. Plasmid linearization.</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104 (50) or 28106 (250)</td>
			<td>Microinjection. For purification of linearized plasmid up to 10 kb.</td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>BP1753I-100</td>
			<td>Microinjection. For purification of linearized plasmid.</td>
		</tr>
		<tr>
			<td>UltraPure Agarose, 500 g</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td>microinjection. Linearized plasmid quantification.</td>
		</tr>
		<tr>
			<td>Nanodrop 2000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/Productnd2000overview.aspx</td>
			<td>microinjection. Linearized plasmid quantification.</td>
		</tr>
		<tr>
			<td>DAPI(4&#39;,6-diamidino-2-phenylindole,dihydrochloride)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td>flow cytometry/FACS</td>
		</tr>
		<tr>
			<td>5 ml polystyrene round bottom tube</td>
			<td>BD Falcon</td>
			<td>352058</td>
			<td>flow cytometry collection tube</td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td>Special Order Research Products (SORP) program</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>5 ml polypropylene round bottom tube</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td>FACS collection tube</td>
		</tr>
		<tr>
			<td>BD LSR II</td>
			<td>BD Biosciences</td>
			<td>Special Order Research Products (SORP) program</td>
			<td>flow cytometry</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, pH 7.4 (1X)</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>FB-01</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Tricaine methanesulphonate (MS-222)</td>
			<td>Western Chemical Inc.</td>
			<td>http://www.wchemical.com/tricaine-s-ms-222.html</td>
			<td>Transportation. Anesthetic. Caution: Irritant. Irritating to eyes, respiratory system, and skin.</td>
		</tr>
		<tr>
			<td>VWR Absorbent Bench Underpads</td>
			<td>VWR</td>
			<td>56616-018</td>
			<td>Transportation. Regular paper towels or sponge can be used as an alternative</td>
		</tr>
		<tr>
			<td>Singe Edge Industrial Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Petri Dish, Polystryrene Disposable Sterile</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Cell Strainer, 40 &#181;m Nylon</td>
			<td>Falcon-Corning Incorporated</td>
			<td>352340</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Corning Incorporated</td>
			<td>430828</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Hemacytometer Set&#160;</td>
			<td>Hausser Scientific</td>
			<td>1483</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Hamilton syringe, fixed needle, volume 10 &#181;L, needle size 26s ga (bevel tip)</td>
			<td>Hamilton</td>
			<td>80366</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Austin&#39;s A-1 Bleach, Commercial</td>
			<td>James Austin Company</td>
			<td></td>
			<td>Transportation. Any commercial&#160; solution can be used</td>
		</tr>
		<tr>
			<td>Ethanol 190 Proof</td>
			<td>Decon Labs, Inc.</td>
			<td>DSP-MD.43</td>
			<td>Microinjection (linearized plasmid purification) and Transportation. Any commercial&#160; solution can be used</td>
		</tr>
		<tr>
			<td>High Speed Microcentrifuge, 300D Digital Microcentrifuge</td>
			<td>Denville Scientific Inc.</td>
			<td>C0265-24</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Sorvall Legend XFR Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004539&#160;</td>
			<td>Transportation. Catalog number for 120V, 60Hz (US)</td>
		</tr>
		<tr>
			<td>5 ml serological pipets</td>
			<td>BD Falcon</td>
			<td>357529</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Corning Stripettor Plus Pipetting Controller</td>
			<td>Corning Incorporated</td>
			<td>4090</td>
			<td>Transportation. Any automatic pipetting controller can be used</td>
		</tr>
		<tr>
			<td>Powder free examination gloves</td>
			<td></td>
			<td></td>
			<td>All steps. Any commercial brand can be used</td>
		</tr>
		<tr>
			<td>Filter pipet tips and micropipettes</td>
			<td></td>
			<td></td>
			<td>All steps. Any commercial brand can be used</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11205-20</td>
			<td>Transportation</td>
		</tr>
		<tr>
			<td>Fluorescent Stereomicroscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td>Scoring. Any appropriate fluorescent stereomicroscope can be used</td>
		</tr>
		<tr>
			<td>Olympus DP72 microscope digital camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>Scoring. Multiple adequate cameras for the selected imaging system can be used.</td>
		</tr>
	</tbody>
</table>

normal and malignant muscle cell transplantation into immune compromised adult zebrafish

斑马鱼已成为一个强大的工具，以评估发育，再生，和癌症。最近，同种异体细胞移植协议已被开发正常和恶性细胞进入照射，同基因和免疫受损的成人斑马鱼的该许可证植入。这些加上优化细胞移植的协议时的模型允许对干细胞功能，再生损伤后，和癌症的快速评估。在这里，我们提出了斑马鱼成人骨骼肌和胚胎性横纹肌肉瘤（ERMS），儿科肉瘤共享功能与胚胎肌肉细胞移植的方法为免疫受损的成人RAG2 E450fs纯合突变的斑马鱼。重要的是，这些动物缺乏T细胞和减少的B细胞的功能，促进大范围从无关供体动物的组织的植入。我们优化的协议显示，荧光标记的肌细胞prepar从α-肌动蛋白RFP转基因斑马鱼ations嫁接强劲时植入的RAG2纯合突变鱼背肌肉。我们也表现出荧光转基因植入ERMS荧光的地方仅限于基于分化状态的细胞。具体地讲，在ERMS AB-应变Myf5的-GFP被创造; mylpfa-mCherry双重转基因动物和肿瘤注射到成年免疫受损鱼腹膜。这些协议的效用延伸到了广泛的正常和恶性供体细胞可被植入到背侧肌肉或成年斑马鱼的腹膜植入。

一种用于制备和α-肌动蛋白的转基因RFP移植供体骨骼肌细胞到免疫受损纯合子突变RAG2斑马鱼 已经证明（协议第1， 图1A和图2）。骨骼肌组织从α肌动蛋白的RFP的转基因供体制备，将所得的单细胞悬浮液含有84.3％的活细胞如以下流式细胞计数分析（ 图2B）评估用DAPI排斥。 RFP阳性细胞包括此单细胞悬浮液（ 图2C）的35.3％。细胞分化成的RAG2纯合突变收件人鱼背侧骨骼肌移植导致一致的和强大的植入所评定分化单细胞向多核纤维（1×10 6个细胞每鱼注射， 表1，图2D-1）。 野生类型接受者 鱼没有嫁接的肌肉纤维在30天的实验（N = 13）。 10天后移植，9出14 RAG2纯合突变的斑马鱼所含注射液（64.3％， 图2E，F）的工地附近的RFP阳性的肌纤维。重要的是，植入RFP阳性肌肉持续至30天移植后（ 图2G-I）中 ，与动物的被跟踪115天后植入和表现出强大的和持久的肌肉植入（数据未示出）的子集。这些结果是类似的那些由我们组23使用相同的协议（ 表1）以前的报告。 我们还介绍了用于产生，制备ERMS肿瘤细胞和移植到腹膜的RAG2纯合突变收件人鱼（协议第2， 图1B和图3）空腔中的方法。 ERMS是在域金字塔之戒产生ê转基因Myf5的-GFP; mylpfa-mCherry鱼已经被证明，以允许肿瘤内异质性和肿瘤细胞亚群移植后11功能分析的可视化。然而，每个亚群的进一步分子表征是困难的，因为鱼是小的时候，他们开发生命的10至30天，肿瘤细胞的数量被限制为下游应用之间ERMS。一种解决方案是通过移植ERMS到成年收件人斑马鱼扩大肿瘤细胞数。迄今为止，类似的实验已经使用CG1应变同基因鱼完成并超过4代回交来开发同基因线条是转基因Myf5的-GFP必需的; mylpfa-mCherry。为了规避这些问题，我们证明免疫受损RAG2纯合突变收件人斑马鱼的实用程序从AB-株嫁接斑马鱼主要ERMS。所有主ERMS植入到RAG2 纯合突变的动物，有利于扩大肿瘤（ 表1）。类似的结果，最近报道，其中的24 27 RAG2纯合突变的斑马鱼嫁接ERMS，而0 7 野生型同胞移植的疾病23。一个嫁接ERMS的代表性例子示在30天时移植后在图3E。嫁接ERMS共享胚胎性横纹肌肉瘤，类似于在原发肿瘤（ 图3B和3F）发现的组织学特征。 FACS分析证实，ERMS载功能不同的肿瘤繁殖表达Myf5的-GFP和/或mylpfa-mCherry细胞和分化的细胞。以下腹腔注射过程存活率超过95％。收件人斑马鱼通常屈服于来自30天移植后的时间点后，肿瘤负荷。 图1.协议示意图（A）的正常和（B）的恶性骨骼肌细胞移植入RAG2纯合突变 斑马鱼可选步骤都标有（*）。 <img alt="图2" src="/files/ftp_upload/52597/52597fig2highres.jpg" width="700px" /> 图2.骨骼肌移植到RAG2纯合突变的斑马鱼。（A）α 肌动蛋白基因RFP捐助斑马鱼。所评定的DAPI染色排除和流式细胞仪（B）的细胞的分离的肌细胞悬液生存力。 （C）的肌细胞悬液中发现的来自α肌动蛋白的RFP给体（红色）RFP阳性细胞的比例，相对于野生型对照（灰色）。 （DE）在30天后移植合并明场和野生动物型 （D）或RAG2纯合突变鱼（E）的荧光图像。 （F）植活率随着时间的推移。红色表示植入动物的数量，而灰色显示非嫁接鱼。指示动物在各时间点分析的次数。 （GI）盒装区域在图E的高倍率图像10（G）所示，图20（H）和30（I）的天移植后，显示出保留了分化的肌纤维随着时间的推移（箭头）。比例尺等于2mm左右。 <a href="https://www.jove.com/files/ftp_upload/52597/52597fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52597/52597fig3highres.jpg" width="700px" /> 图3.移植Myf5的-GFP的; mylpfa-mCherry ERMS到RAG2纯合突变 。斑马鱼（AD）RAG2-KRASG12D引起的原发于AB应变Myf5的-GFP所产生的ERMS; mylpfa-mCherry斑马鱼生活在30天。 （EH）RAG2纯合突变斑马鱼嫁接了ERMS和30天移植后进行分析。 （A，E）合并的明场和初级和移植ERMS的荧光图像。肿瘤区域概述和箭头指示注射部位在电子商务。 （B，F），苏木和主（B）的曙红染色的石蜡切片和嫁接ERMS（F）中示出与癌症相关的增加的细胞构成的区域。 （C，作为评估DAPI染色排除和流式细胞仪G）细胞活力。 （D，H）荧光的肿瘤细胞亚群，如评估通过流式细胞术。比例尺等于2毫米（A，E）和50微米（B，F）。 <a href="https://www.jove.com/files/ftp_upload/52597/52597fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表1" src="/files/ftp_upload/52597/52597table1.jpg" width="700px" /> 表1.植活的结果为肌肉和ERMS细胞移植。（*）表示以往报告的数据使用相同的技术23。数据转载自自然法的许可。 <a href="https://www.jove.com/files/ftp_upload/52597/52597table1.jpg" target="_blank">请点击这里查看此表的放大版本。</a>。

Watch this Scientific Journal Video about Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish at JoVE.com

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

正常和恶性肌细胞移植到免疫受损成年斑马鱼

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

normal-malignant-muscle-cell-transplantation-into-immune-compromised

Research

JoVE Journal

Immunology and Infection

10.8K 次观看.  Massachusetts General Hospital. Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells. 

视频: Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

成年斑马鱼的口服插管: 一种评价生物活性化合物肠道吸收的模型

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...