Name: using zebrafish models of human influenza a virus infections to screen antiviral drugs and characterize host immune cell responses
Uploaded: 2017-01-20T15:00:00.000+00:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses at JoVE.com

The authors wish to thank Mark Nilan for zebrafish care and maintenance and Meghan Breitbach and Deborah Bouchard for propagating NS1-GFP and determining IAV titers. This research was supported by NIGMS grant NIH P20GM103534 and the Maine Agricultural and Forest Experiment Station (Publication Number 3493).

所有工作都应该使用生物安全2级（BSL2或）由美国疾病控制中心（CDC），并按照机构动物护理和使用委员会（IACUC）制定的指令所描述的标准来执行。请有关官员商议，以确保安全和法规遵从。 1.斑马鱼保养与维护<ol><li>斑马鱼产卵，并收集所需数量的胚胎实验。必要时，大规模繁殖罐，像那些由Adatto 等人描述。 32，可用于收集大量的发育上演胚胎。 </li><li>允许胚胎发展到在低密度深培养皿中所需的发育阶段（48小时受精后的全身IAV感染[第3或5天受精后的本地化，鱼鳔感染[第4]）（&lt;含6无菌鸡蛋水100胚胎/皿）在28°C0微克/毫升海盐（ 例如 ，即时海洋）在蒸馏水。 <ol><li>用塑料吸管转移去除死胚胎和卵子变化，每天的水，以保证最佳的健康和发展。 </li></ol></li></ol> 2.材料和试剂的制备<ol><li>制备4毫克/毫升的库存三卡因-S的蒸馏水和高压釜溶液（调节pH值至7.0-7.4）。保存在4℃。 </li><li>用一个微量拉马长丝拉硼硅玻璃毛细管（ 例如微管普勒使用以下设置:压力设定= 100，热量= 550，拉= [无值]，速度= 130，时间= 110）。 注意:在此之前修整所述针，从那里的针开始逐渐变细，以在针的末端的长度应约为12-15毫米。然而，这可能会有所不同，根据自己的偏好和应用。更长，更渐进的锥度可能因为刺破胚胎增加的能力和降低RI更佳针弯曲或断裂的SK。 </li><li>消融2 g用微波在100毫升水蛋琼脂糖。使在其上排队并通过倾琼脂糖熔化成深培养皿中并使其固化注入胚胎板。 </li><li>使扎那米韦的10毫克/毫升的库存在无核酸酶的水。分装，冷冻在-20℃。 </li><li>利用微波使胚胎安装琼脂糖融化在50毫升水蛋0.5克琼脂糖。维持水浴在50℃下，直到准备成像期间使用。 </li></ol> 3.全身感染IAV（48小时受精后） 注意:所有的研究人员应与上级和医生就在开始工作之前与IAV疫苗接种咨询。 <ol><li>手动dechorionate使用＃5钳实验当天胚胎。小心取出，安乐死已经在200微克/毫升三卡因溶液的过程中受伤的胚胎。 </li><li> Ø准备˚FIAV显微注射 注:株已被证明感染斑马鱼胚胎是流感A / PR / 8/34（H1N1）（APR8），甲型流感的X 31 A /爱知县/ 68（H3N2）（X-31），以及荧光记者流感病毒NS1-GFP 33。有关APR8和X-31和其他菌株的详细信息，可以在流感研究数据库中找到<ol><li>传播IAV的NS1-GFP株鸡胚如前所述34,35。收集，分装和滴定含有IAV尿囊液，并储存在-80℃。预滴定APR8和X-31的病毒可以商业购买。 <ol><li>只是在感染前，迅速解冻的IAV等分在戴手套的手，然后迅速置于冰上减少滴度的损失。 </li></ol></li><li>病毒稀释<ol><li>如果使用的是APR8或X-31病毒，稀释至3.3×10 6个蛋感染剂量50％（EID 50）/ ml，在无菌的，冰冷的PBS supplemented用0.25％酚红在层流罩（在可视化助剂）。同时建立包含无菌PBS，0.25％酚红控制。 </li><li>如果使用NS1-GFP应变33，稀释至〜1.5×10 2噬菌斑形成单位（PFU）每NL在无菌的，冰冷的PBS含有0.25％酚红（在可视化助剂）在层流罩。同时建立含稀释就像在无菌PBS病毒与0.25％酚红未被感染的鸡蛋尿囊液控制。 </li><li>维持IAV冰上待用。 </li></ol></li><li>吸取病毒解决方案为使用微加载提示，使用前显微注射针。插入注射针头插入注射装置（ 例如 ，移动电话倡议-3-压喷射系统）的适当支架。 </li><li>在观看立体显微镜下，轻轻一夹有棱，消毒＃5镊子显微注射针头。 注:这是recommenDED的显微注射针尖逐渐裁剪。 </li><li>压低的压力喷射系统的脚踏板和注入显微镜浸油上的校准千分尺滑下降。测量液滴的直径。计算下降使用公式的体积，V = 4 /3πr3，其中V是体积在n1和r是在微米的半径。 注意:如果放置直径太小，附加玻璃可以被裁剪或压力和时序设置可调节。如果下降直径太大，压力和时序设置可调节。 </li><li>上的压力喷射器调节压力和时序设置和/或直至所需的注射体积达到（一般为1-3 NL）reclip针尖。 40和80毫秒之间的20和30 PSI和脉冲宽度设置之间的压力设置是典型的。调整回压，使其专柜毛细管压力，但不漏IAV（净零压力）。 </li></ol></li><li>对齐胚胎注射<ol><li>在200微克/毫升三卡因麻醉dechorionated胚胎。 </li><li>一旦运动停止，转移10-20胚胎至2％的琼脂糖平板（第2.3节中的方法制备）用塑料移液管。用塑料吸管以除去过量的液体。 </li><li>轻轻对准胚胎与火抛光和密封的硼硅玻璃毛细管注射。使用立体显微镜，轻轻东方胚胎以便居维叶的管道或后部主静脉与所述注射针（ 图1A）线。 </li></ol></li><li> IAV显微注射<ol><li>轻轻地插入注射针头插入居维叶管道或后主静脉。踩下脚踏注入IAV的所需体积进入胚胎（ 图1A）的循环系统。胚胎如有必要，可注入的一个以上的时间。 注意:注射小丸应该被扫成流通和DISTRIB整个身体的布式。如果注射体积收集在注射部位，从实验和安乐死移除胚胎。 </li><li>重复注射在不同的胚胎具有特定病毒所选择的应变控制解决方案。对于APR8和X-31，使用3.2.2.1节描述的控制;为NS1-GFP，使用3.2.2.2节描述的控制。 </li></ol></li><li>胚胎转移到含有无菌恒温箱鸡蛋水和地方在33°C标记的培养皿。 注:感染胚胎应在33℃下生长，以支持增强的病毒复制。另外，培养在33℃下更紧密地模仿人上呼吸道，这是在外部环境中的具有较低温度的紧密接触，并且在那里IAV感染发生的温度。胚胎可以适应于广泛的温度范围内34。 </li></ol> 4.本地化，鱼鳔IAV感染的Tg（MPX:mCherry） </EM&gt;转基因斑马鱼（5天受精后） <ol><li> IAV的制备显微注射<ol><li>如第3.2节所述稀释NS1-GFP应变和控制注射液。 </li><li>如在2.2节所述准备针。 </li><li>对齐的Tg（MPX:mCherry）含有与以下异常3.3节所述膨胀鱼鳔35幼虫。以这样的方式使针可穿透鱼鳔和病毒可以存入鱼鳔的后部，如前所述36（ 图2A）对齐幼虫。 </li></ol></li><li> IAV显微注射<ol><li>轻轻插入注射针头插入鱼鳔并朝向结构（ 图2A）的后注入所需量（ 例如 5 NL）。 注:注射小丸应收集朝着后和鱼鳔的气泡应该bË向前移位。 GFP表达应该开始在3小时后注射待观察。 </li><li>具体到病毒的选择，如3.4.2节中描述的应变控制解决方案，重复注射。 </li></ol></li><li>幼虫转移到含有无菌恒温箱鸡蛋和水发生在33°C的培养皿。 </li></ol> 5.抗病毒治疗药物注意:以下协议描述以前的Gabor 等所示的扎那米韦治疗。 19。该协议可以被修改以筛选其它抗病毒药物，并具有进行修改，以筛选在96孔板，高通量格式多种化合物的潜力。 <ol><li>在3小时后感染，用含0，16.7，33.3纳克/毫升无菌扎那米韦鸡蛋水代替NS1-GFP-的鸡蛋水和控制感染的鱼。 注意:这些浓度才能被选为试图复制FO达到的生理水平在人类患者中（100，200，或600毫克，四，每日两次或10mg吸入，每天两次）扎那米韦的llowing施用。在人类患者血清浓度范围为9.83至45.3纳克/毫升36。 </li><li>在5天的过程中，改变蛋水每12小时，并用含有扎那米韦适量无菌蛋水替换。 </li><li>追踪感染的课程。 <ol><li>在200微克/毫升三卡因麻醉斑马鱼。监测立体荧光显微镜，目视观察IAV感染和控制之间的鱼，而鱼浸泡在不同浓度的药物中的感染模式的差异GFP表达。 </li><li>跟踪发病率和死亡率在5天的过程中。 <ol><li>有关与疾病的病理感染的动态，包括嗜睡的症状和水肿的证据，色素沉着的差异，眼和颅面畸形，以及前凸记录观察结果。疾病的病理通常在24-48小时后喷射成为控制感染鱼显而易见，这取决于病毒的注入量。采集图像后24小时注射。 </li><li> 5天感染后每隔24小时，记录死亡，病态和健康幼虫的数目。死亡是由缺乏明显的心跳的定义。根据需要绘制的数据。例如，绘图数据作为堆积条形图，用健康，病态，或死鱼百分比在y轴和治疗组在x轴上。 19 注:死亡可能由Kaplan-Meier生存估计值进行评分。根据应用，也可适当开发用于评分鱼发病的各种途径，包括可能量化基于所观察到的上述病理特征的程度的发病率的评分矩阵。 </li><li>与统计学家咨询，以申请相应的统计分析。 </li></ol></li></ol></li></ol> 6。中性粒细胞迁移注:下面的协议描述用于跟踪中性粒细胞迁移到鱼鳔以下本地化，上皮IAV感染的方法。描述的方法可以被修改，以测试遗传和化学处理的效果。使用这种技术，将有可能在IAV感染期间表征底层嗜中性粒细胞的行为的机制。 <ol><li>安装斑马鱼成像<ol><li>麻醉幼虫在200微克/毫升三卡因进行成像。 </li><li>鸡蛋中的水（〜50-100微升）的一小滴已经感染了NS1-GFP到24的好啦幼虫，玻璃底板:传送单个的Tg（MPX mCherry）。与其他IAV感染和控制鱼重复。 </li><li>慢慢的1％琼脂糖胚胎的安装介质添加到每口井（2.5节），注意不要引入大泡。 <ol><li>这样它被安装在其一侧轻轻调整幼虫的位置。古迪和亨利37描述如何使使用被安装成与长丝硼硅玻璃毛细管和代替强力胶水粘上昆虫针在本申请中运作良好探针。 </li></ol></li><li>一旦琼脂糖变硬，轻轻地用含有200微克/毫升三卡因鸡蛋水填满个别井。 </li><li>用共聚焦显微镜，拍摄AZ系列堆栈使用20倍的目标明和荧光图像的集中在鱼鳔。 <ol><li>使得它们捕获所有明显可观察到的绿色和红色荧光设置上限和下限。 </li><li>在2.0微秒/像素的拍摄图像与那些≤4微米的步骤。增加时间每像素和还原步骤之间的距离（ 例如 ，0.5 - 1.0微米）增加图像的分辨率和质量。 </li><li>产生通过合并的层的二维图像。 </li><li>比较MPX-mCherry阳性嗜中性粒细胞目前我的数ñIAV感染和控制注入​​斑马鱼的鱼鳔。 </li></ol></li></ol></li></ol>

The authors have nothing to disclose.

为了最大限度地使用小动物到人类宿主 - 病原体相互作用的模型获得的利益，框架研究问题，并在测试假说，关于模型系统的固有优势利用是很重要的。作为人类IAV感染模型，斑马鱼有几个优势，包括高繁殖力，光学清晰度，顺从于药物筛选，以及该标签的免疫细胞嗜中性粒细胞一样的转基因株系的可用性。斑马鱼已经发展成为日益强大的替代鼠标模型系统的炎症和先天免疫方面的研究。因为他?...

据世界卫生组织（WHO），流感病毒感染的成年人的5％-10％，每年儿童的20-30％，并导致300万-500万严重病例和全世界1到500,000人死亡。对流感疫苗接种每年仍然以防止疾病的最佳选择。像世卫组织全球行动计划的努力，以降低发病率和季节性流感暴发2相关的死亡率增加了季节性流感疫苗的使用，疫苗生产能力，以及研究和开发更有效的疫苗策略。抗病毒药物像神经氨酸酶抑制剂（ 例如 ，扎那米韦和奥司他韦）是在一些国家可用以及缓解症状已被证明有效，当发病3，4，5的第一48小时内给药。尽管全球努力，季节性流感遏制欧tbreaks保持此时一个严峻的挑战，因为流感病毒抗原漂移往往超过适应病毒6的改变基因组中的电流的能力。疫苗策略靶向病毒的新菌株必须提前开发，有时由于在类型菌株，在一个流感季节最终占优势不可预见的变化呈现小于最佳效果。由于这些原因，有一个明确需要制定包含感染和死亡率降低替代治疗策略。通过实现更好的理解在主机病毒相互作用，有可能开发新的抗流感药物和辅助疗法7,8。 人宿主A型流感病毒（IAV）相互作用是复杂的。人类IAV感染的几种动物模型为了深入了解宿主病毒相互作用，大型的源码已经开发ING小鼠，豚鼠，棉鼠，仓鼠，雪貂，猕猴和9。同时提供具有增强宿主IAV动力学的理解的重要数据，每个模式生物拥有必须尝试将结果转化为人类医学时，应考虑显著的缺点。例如，小鼠，这是最广泛使用的模型，不容易与人流感分离株9感染时开发IAV诱导感染症状。这是因为小鼠缺乏对人流感的天然趋向性分离自小鼠上皮细胞表达α-2,3-唾液酸连接，而不是对人上皮细胞10表示的α-2,6唾液酸连接。存在于人类IAV的血凝素蛋白的分离从优结合并进入宿主细胞到通过受体介导的内吞作用9，11α-2,6唾液酸连接， </s达&gt; 12，13。因此，现在已被接受，在发育的小鼠模型的人流感，必须小心，以与流感的适当应变配对鼠标的适当应变，以达到疾病表型概括的人类疾病的各个方面。与此相反，在雪貂的上呼吸道上皮细胞具有类似于人类细胞14α-2,6唾液酸连接。感染雪貂共享许多在人类疾病中观察到的病理和临床特征，包括人类和禽流感病毒14的致病性和透射性，15。它们也是高度适合于疫苗功效试验。然而，对于人类流感鼬模型有几个缺点，主要与它们的大小和饲养成本，使统计上显的收购具有挑战性着数据。此外，雪貂以前已经显示在药代动力学，生物利用度和毒性，使测试功效很难区别。例如，雪貂表现出毒性M2离子通道抑制剂金刚烷胺16。因此，很显然，在选择的动物模型来研究有关人类IAV感染的问题，必须考虑其固有的优点和局限性，并在主机病毒相互作用是受调查的方面是非常重要的。 斑马鱼， 斑马鱼是动物模型，提供了独特的机会，研究微生物感染，宿主免疫反应，和潜在的药物治疗17，18，19，20，21，22，23，&lt;SUP类="外部参照"&gt; 24，25，26，27，28。 α-2,6-连接的唾液酸在斑马鱼细胞的表面上存在表明其易感性IAV，其在感染的研究证明了，用IAV 19的荧光报道菌株在体内成像。在IAV感染斑马鱼，抗病毒ifnphi1和MXA转录物的表达增加表明先天免疫应答已经刺激，并通过IAV感染斑马鱼，包括水肿和组织破坏显示的病理，是类似于在人类流感感染观察。此外，IAV抗病毒药神经氨酸酶抑制剂扎那米韦有限死亡率和在斑马鱼19减少病毒复制。 在这份报告中，对于引发体系协议在斑马鱼胚胎IC IAV感染描述。使用扎那米韦临床相应剂量的作为证明的原则，对于抗病毒活性的化合物筛选斑马鱼这种IAV感染模型的效用是证明。此外，用于产生局部的，上皮IAV感染在斑马鱼的协议鱼鳔，被认为是在解剖学和功能上类似于哺乳动物的肺21，29，30，31之间的一个器官，进行说明。使用这种局部IAV感染模型，中性粒细胞募集至感染部位可以跟踪，使调查中性粒细胞生物学在IAV感染和炎症中的作用。这些斑马鱼模型补充人类IAV感染的现有的动物模型，并且用于测试的小分子和由于增强了S的可能性免疫细胞应答特别有用tatistical功率，容量为中到高通量测定法，和能力来跟踪免疫细胞的行为和功能的光显微镜。

<table><tbody><tr><td>Instant Ocean</td><td>Spectrum Brands</td><td>SS15-10</td><td></td></tr><tr><td>100 mm x 25 mm sterile disposable Petri dishes&amp;nbsp;</td><td>VWR</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes&amp;nbsp;</td><td>Fisherbrand</td><td>13-711-7M</td><td></td></tr><tr><td>Tricaine-S (MS-222)</td><td>Western Chemical</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary with filament&amp;nbsp;</td><td>Sutter Instrument&amp;nbsp;</td><td>BF120-69-10</td><td></td></tr><tr><td>Flaming/Brown micropipette puller&amp;nbsp;</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Zanamivir</td><td>AK Scientific</td><td>G939</td><td></td></tr><tr><td>Dumont #5 forceps&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>72700-D</td><td></td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Microscope immersion oil</td><td>Olympus</td><td>IMMOIL-F30CC</td><td></td></tr><tr><td>Microscope stage calibration slide&amp;nbsp;</td><td>AmScope</td><td>MR095</td><td></td></tr><tr><td>MPPI-3 pressure injector&amp;nbsp;</td><td>Applied Scientific Instrumentation</td><td></td><td></td></tr><tr><td>Stereo microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Phenol red&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P-4758</td><td></td></tr><tr><td>Low temperature incubator</td><td>VWR</td><td>2020</td><td></td></tr><tr><td>SteREO Discovery.V12</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Illuminator</td><td>Zeiss</td><td>HXP 200C</td><td></td></tr><tr><td>Cold light source</td><td>Zeiss&amp;nbsp;</td><td>CL6000 LED</td><td></td></tr><tr><td>Glass-bottom multiwell plate, 24 well</td><td>Mattek</td><td>P24G-0-13-F</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>Fluoview software</td><td>Olympus</td><td></td><td></td></tr><tr><td>Prism v6</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Influenza A/PR/8/34 (H1N1) virus&amp;nbsp;</td><td>Charles River&amp;nbsp;</td><td>490710</td><td></td></tr><tr><td>Influenza A X-31, A/Aichi/68 (H3N2)&amp;nbsp;</td><td>Charles River&amp;nbsp;</td><td>490715</td><td></td></tr><tr><td>Influenza NS1-GFP</td><td></td><td></td><td>Referenced in Manicassamy &lt;em&gt;et al.&lt;/em&gt; 2010</td></tr><tr><td>Tg(mpx:mCherry)</td><td></td><td></td><td>Referenced in Lam &lt;em&gt;et al.&lt;/em&gt; 2013</td></tr></tbody></table>

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.

这里，提供了示出如何全身IAV感染在斑马鱼可用于测试药物功效（ 图1A）的数据。在48小时后的受精胚胎经由居维叶的导管与APR8（ 图1C，1F）中，X-31（ 图1D，1G），或NS1-GFP（ 图1 H-1I）注入以引发病毒感染。在48小时后的受精胚胎另一个队列注射，作为病毒感染（ 图1B，1E）控制。通过48小时后感染，斑马鱼注?...

Watch this Scientific Journal Video about Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses at JoVE.com

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

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 ...

using-zebrafish-models-human-influenza-virus-infections-to-screen

Research

JoVE Journal

Immunology and Infection

9.8K 次观看.  University of Maine. 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. 

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

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

科研

癌症研究

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

免疫与感染

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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 ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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

摘要

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此视频中的章节

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

建立和高通量安装程序研究优化

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

先天免疫反应中的斑马鱼幼体型号无创成像

流感病毒在小鼠感染模型中的研究

marinum 分枝杆菌感染成人斑马鱼结核的建模

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

在体内病态的人类基因组建模使用斑马鱼

手册小分子屏幕接近高通量使用斑马鱼胚胎

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

摘要

探索更多视频

此视频中的章节

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

建立和高通量安装程序研究优化

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

先天免疫反应中的斑马鱼幼体型号无创成像

流感病毒在小鼠感染模型中的研究

marinum 分枝杆菌感染成人斑马鱼结核的建模

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

在体内病态的人类基因组建模使用斑马鱼

手册小分子屏幕接近高通量使用斑马鱼胚胎

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用