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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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><tbody><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&amp;nbsp;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&#039;,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>transplantation</td></tr><tr><td>Fetal Bovine Serum</td><td>Omega Scientific</td><td>FB-01</td><td>transplantation</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>Transplantation. 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>Transplantation. 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>transplantation</td></tr><tr><td>Petri Dish, Polystryrene Disposable Sterile</td><td>VWR</td><td>25384-302</td><td>transplantation</td></tr><tr><td>Cell Strainer, 40 &amp;micro;m Nylon</td><td>Falcon-Corning Incorporated</td><td>352340</td><td>transplantation</td></tr><tr><td>50 ml Centrifuge Tube</td><td>Corning Incorporated</td><td>430828</td><td>transplantation</td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Life Technologies</td><td>15250-061</td><td>transplantation</td></tr><tr><td>Hemacytometer Set&amp;nbsp;</td><td>Hausser Scientific</td><td>1483</td><td>transplantation</td></tr><tr><td>Hamilton syringe, fixed needle, volume 10 &amp;micro;l, needle size 26s ga (bevel tip)</td><td>Hamilton</td><td>80366</td><td>transplantation</td></tr><tr><td>Austin&#039;s A-1 Bleach, Commercial</td><td>James Austin Company</td><td></td><td>Transplantation. Any commercial 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 transplantation. Any commercial&amp;nbsp; 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>transplantation</td></tr><tr><td>Sorvall Legend XFR Centrifuge</td><td>Thermo Scientific</td><td>75004539&amp;nbsp;</td><td>Transplantation. Catalog number for 120 V, 60 Hz (US).</td></tr><tr><td>5 ml serological pipets</td><td>BD Falcon</td><td>357529</td><td>transplantation</td></tr><tr><td>Corning Stripettor Plus Pipetting Controller</td><td>Corning Incorporated</td><td>4090</td><td>Transplantation. 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>transplantation</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.9K 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

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 Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

癌症研究

利用斑马鱼幼虫进行高效的体内肿瘤研究

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

作者聚焦:利用斑马鱼肿瘤异种移植推进癌症治疗

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

<meta charset="utf8"></head><body>使用细胞增殖染料分选估计斑马鱼 T-ALL 中的 LSC 频率

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

<meta charset="utf8"></head><body>作者聚焦:从斑马鱼 T-ALL 中鉴定和分离静止白血病干细胞

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞 

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

发育生物学

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

正常和恶性肌细胞移植到免疫受损成年斑马鱼

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在成年斑马鱼的全肾骨髓移植

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

一种简单有效的斑马鱼胚胎移植装置

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼