我们衷心感谢重庆医科大学人才项目（项目编号:R4014，项目编号:R4020）、国家自然科学基金项目（国家自然科学基金项目，编号:32200386）、重庆市博士后创新导师工作室（X7928 DSP）、中国科学院中斯水技术研究示范联合中心项目/中科院中斯联合教育研究中心等项目的支持。

鱼类实验是按照重庆市动物护理和使用专业委员会和重庆医科大学动物护理和使用机构的指导原则以及国家质量技术监督局颁布的实验动物标准（批准号:GB14922-2001-GBT14927-2001）进行的。斑马鱼（Danio rerio，野生型，AB品系）来源于中国科学院水生生物研究所，并按照先前报道的程序在实验室中保存36。
1.成年斑马鱼的维护和胚胎采集
注:借鉴先前研究和报告16,37,38,39的修改，我们实验室中成年斑马鱼的维持，幼虫饲养和无菌（GF）模型的实践是按照下面概述的步骤进行的。用于鱼类养殖的超纯流量系统已获得商业化（见材料表）。水在保持平衡的 pH 值、盐和碱的情况下，在循环过程中经过过滤以确保清洁条件。
<ol><li>在自动循环系统中按照以下标准程序（见 材料表）保持成年鱼，光周期为暗:光 = 10 小时:在 28 °C ± 0.5 °C 下 14 小时，并用新鲜孵育的 卤虫 属喂养。 每日两次。</li>
	<li>前一天晚上将性成熟的成年斑马鱼（雄性:雌性 = 2:1）放入装有新鲜干净水的水箱中。</li>
	<li>让鱼在第二天早上（大约上午8:30）在实验室的常规光刺激下产卵。1-2小时后，将鱼转移到另一个鱼缸中。</li>
	<li>使用一次性巴斯德移液管将胚胎从自动循环系统收集到水中作为培养基在培养皿中。</li>
</ol>2. GF斑马鱼从胚胎到幼虫的生成
注: 图1概述了产生无菌（GF）鱼的程序，而传统（CR）和GF模型的基本发育指标则在 补充图1中显示。请在洁净室中使用无菌技术在台式生物安全柜或层流罩中按照下面概述的步骤进行操作。
<ol><li>用鱼培养水清洗胚胎，去除粪便、杂质和死卵或未受精卵。</li>
	<li>将胚胎以2hpf转移到装有抗生素无菌斑马鱼培养基（AB-GZM，参见 材料表）溶液的灭菌板（每个无菌培养皿最多480个胚胎，直径为10cm）溶液，并在28.5°C下培养6-8小时。 注意:AB-GZM用于处理胚胎数小时以消除表面微生物，而不含抗生素的GZM用于在亚序列中培养GF鱼。通常，完美的培养时间为6小时）。</li>
	<li>排除有白色斑点或白色外观的鸡蛋，并选择那些正常发育的鸡蛋，显示透明和完整的包膜以供进一步加工。</li>
	<li>在10分钟内使用AB-GZM冲洗胚胎三次。</li>
	<li>将胚胎浸泡在 0.2 g/L 聚维酮碘溶液 （PVP-I） 中 1 分钟（参见 材料表）。 注意:更高的浓度和超过 2 分钟的时间会影响胚胎的死亡和孵化率。</li>
	<li>在10分钟内使用无菌斑马鱼培养基（GZM）洗涤胚胎三次。</li>
	<li>将胚胎加入次氯酸钠（NaClO）工作溶液中，紧紧覆盖50mL微量离心管，漂白15分钟。在此期间，轻轻摇晃胚胎，将胚胎悬浮在漂白剂溶液中。</li>
	<li>在10分钟内使用GZM洗涤胚胎三次。</li>
	<li>将胚胎转移到无菌的 6 孔板中，每孔有 5 个胚胎和 10 mL GZM。在光周期为暗的超洁净平台或培养箱中培养它们:光 = 10 小时:28 °C ± 0.5 °C 下 14 小时。</li>
	<li>每天通过去除用过的 GZM 来更新培养基，将其收集为用于无菌状态检测的样品，并用相同体积的无菌 GZM 补充每个孔。</li>
	<li>记录GF胚胎/幼虫的基本发育指标，包括存活率、孵化率、心跳、体长和体重等19。</li>
	<li>将GF鱼转移到合适的容器（培养皿，细胞培养瓶或带盖的玻璃瓶）中，以扩大体积，在整个生长过程中提供无菌食物。</li>
</ol>3. GF斑马鱼从幼虫到幼鱼的维护
<ol><li>在 7 dpf 之前不进食的情况下更新 GZM，并每天检测无菌状态（参见步骤 5）。</li>
	<li>在更换GZM之前，每天一次用无菌蛋黄（每孔10μL制备的储备溶液）喂养8 dpf至幼体阶段的斑马鱼幼虫。</li>
	<li>用灭菌蛋黄与GF 卤虫 混合喂养GF幼鱼，每天一次（1-3只 卤虫/幼虫，见步骤4），随着GF鱼的生长和发育逐渐增加食物量。</li>
	<li>提供蛋黄，直到所有鱼都可以食用 卤虫无节幼体。 注意:GF 卤虫 应在使用前进行无菌测试。评估剩余的 卤虫无节幼 体数量，观察追捕和捕捉进度，并检查含有食物的鱼体以指示摄食成功。</li>
	<li>从28 dpf到早期成虫阶段，每天喂食一次GF 卤虫 （3-5只 卤虫/幼虫），并清理未开发或死亡的 卤虫，以及死鱼作为每日样本进入废弃的GZM中。</li>
	<li>在GF鱼生长期间，将7 dpf后的6孔板更改为无菌板或细胞培养瓶，从8dpf更改为21dpf，然后在21dpf后更改为无菌瓶。根据每个容器中GF鱼的数量和重量增加培养基和食物质量。</li>
	<li>每天更新GZM并检查样品，以确保GF模型的成功。 注意:在成年早期和性成熟之前保持无菌（GF）鱼类模型具有挑战性，平均存活率低至30 dpf，通常低于10%。这主要归因于免疫力低下、发育迟缓和肠道功能受损。</li>
</ol>4. GF卤虫无节幼体作为慢性GF鱼模型不育食品的制备
注:无菌（GF） 卤虫的 培养和制备方法如图 2所示。请注意，以下所有步骤均在台式生物安全柜中使用无菌技术进行。
<ol><li>用 2.4 g/L 次氯酸钠（NaClO，参见材料表）冲洗 0.25 g 卤虫囊肿 5 分钟。</li>
	<li>用灭菌的超致密过滤器（孔径约170目）过滤冲洗过的 卤虫 囊肿，并用灭菌的超纯水洗涤三次，每次1-2分钟。</li>
	<li>将洗涤过的 卤虫 囊肿转移到 100 mL 2.5% 灭菌盐水中，混合并按照下面提到的步骤包装用于无菌孵化。</li>
	<li>将50mL处理 的卤虫 囊肿混合溶液装入体积为100mL的无菌瓶中，用薄膜密封，并在150rpm，30°C的振荡培养箱中用光孵育18小时至24小时。</li>
	<li>使用一次性巴斯德移液管从中间层和下层提取约 30 mL 孵化 卤虫无节幼 体。 注意: 防止漂浮的鸡蛋和空壳到达上层。无菌（GF） 卤虫 囊肿和 无节幼 体孵育24小时的指标如图 3所示。</li>
	<li>用灭菌过滤器过滤 卤虫 ，并在灭菌烧杯中用灭菌超纯水洗涤它们三次，每次约30秒至1分钟。最后，加入 4 mL 灭菌超纯水以制备 卤虫 混合溶液。</li>
	<li>使用一次性巴斯德移液管，从上层取出活跃游泳的 卤虫 ，洗涤并准备 无节幼 体溶液用于喂养和无菌鉴定。</li>
</ol>5. GF鱼全生命周期模型的鉴定
注:在无菌（GF）鱼的整个生命周期阶段，关键指标和每日样本检测对于确保模型的成功至关重要。此步骤总结了样品的常见分类和常用的鉴定方法（图4）。
<ol><li>每天观察并统计GF斑马鱼幼虫的存活状况和率。在计算相关速率的实验中，GF鱼在24孔或48孔板中养殖，每孔一条鱼。<ol><li>成活率=（观察时活鱼数/卵总数）×100%。</li>
		<li>不育率=（不育鱼数/存活鱼数）×100%。 注:该协议侧重于活鱼的不育率。不育鱼的数量是通过在活鱼中多次检查后计算成功的GF鱼来确定的。任何因细菌存在而失败的死鱼或活鱼 GF 模型都会在随后的 GF 程序中被消除。</li>
	</ol></li>
	<li>收集以下GF斑马鱼幼虫样本。<ol><li>来自每个孔或瓶子容器的GF斑马鱼培养基。</li>
		<li>鱼食，如无菌蛋黄和GF 卤虫。</li>
		<li>废物介质，包括孵化后的卵膜碎片和幼鱼到幼鱼在摄食期间的食物残渣或粪便。</li>
		<li>死鱼样本，用于识别细菌的存在。</li>
		<li>用于胚胎处理的鱼培养基和试剂作为无菌对照。</li>
		<li>来自材料、操作平台和培养箱等的样品</li>
	</ol></li>
	<li>使用多种方法检测每天收集的样本。<ol><li>首先，使用铺展板法并在30°C的培养箱中培养板。<ol><li>在有氧条件下，将 20-50 μL 废培养基样品涂在胰蛋白酶大豆琼脂 （TSA） 板上（参见 材料表）。</li>
			<li>在厌氧条件下，在 TSA 板上涂有 20-50 μL 废培养基样品。</li>
			<li>在有氧条件下，将 20-50 μL 废培养基样品涂在血液 TSA 板上（参见 材料表）。</li>
			<li>在厌氧条件下将 20-50 μL 废培养基样品涂在血液 TSA 板上。</li>
		</ol></li>
		<li>第二，采用液体培养法。<ol><li>将 200 μL 样品放入装有脑心输液 （BHI） 培养基（参见 材料表）的需氧管中，并在 30 °C、150-180 rpm 的振荡培养箱中培养。</li>
			<li>将200μL样品加入装有BHI培养基的厌氧管中，并在30°C的培养箱中培养。</li>
			<li>将 200 μL 样品加入装有胰蛋白酶大豆肉汤 （TSB） 培养基的需氧管中，然后在 30 °C、150-180 rpm 的振荡培养箱中培养。</li>
			<li>将 200 μL 样品加入装有 TSB 培养基的厌氧管中，并在 30 °C 的培养箱中培养。</li>
		</ol></li>
		<li>第三，采用分子技术-16s聚合酶链反应（PCR）测定。<ol><li>用两对引物 27F 和 1492R 以及 63F 和 1387R 分析废水样品（表 1）。 注:PCR预混液是根据 表2 使用市售的DNA聚合酶试剂盒（参见 材料表）制备的，PCR机的设置程序为表 3中指定的程序。</li>
			<li>PCR程序完成后，通过1%琼脂糖凝胶电泳40 在1465bp（使用引物27F和1492R）或1324bp（使用引物63F和1387R）的目标条带上检查产物，以推断样品的无菌性。</li>
		</ol></li>
	</ol></li>
	<li>记录并观察无菌测试，包括24小时至3天和7天的平板和培养基培养，在此期间，平板上没有菌落，液体中没有浊度，表明GF模型的成功构建。在24小时，48小时，72小时，96小时，120小时，144小时和168小时观察并记录板和培养基。</li>
</ol>6. 在以GF鱼为食之前鉴定GF 卤虫 样本
注意:要检测GF 卤虫 样本，在喂养GF鱼之前作为活食是必不可少的（图4），请按照以下步骤操作。
<ol><li>收集 GF 卤虫 模型样本。<ol><li>未经治疗 的卤虫 囊肿。</li>
		<li>治疗过GF 卤虫 囊肿。</li>
		<li>孵化的 GF卤虫无节幼体。</li>
		<li>灭菌超纯水作为对照。</li>
	</ol></li>
	<li>使用以下方法检测GF 卤虫 。<ol><li>在有氧和厌氧条件下，使用扩散板法通过将它们涂覆在 TSA 板和血液 TSA 板上来检测样品。将它们在30°C下孵育。</li>
		<li>使用液体培养基检测好氧和厌氧 TSB 和 BHI 管中的样品。在振荡器中以150-180rpm，30°C培养。</li>
		<li>使用PCR测定法检测使用16s核糖体RNA靶基因引物27F和1492R，63F和1387R收集的样品中是否存在细菌（表1）。音量和程序如 表2 和 表3所示。</li>
	</ol></li>
	<li>记录结果:通过1%琼脂糖凝胶电泳40 检测PCR产物，用于测量1465 bp（使用引物27F和1492R）或1324 bp（使用引物63F和1387R）的靶带。平板和液体培养基的方法可以保证 GF卤虫 在用作GF鱼食前的无菌状态。</li>
</ol>7. 斑马鱼肠道细菌菌株的分离鉴定
注:为了探索 体外 和 体内的肠道功能，有必要从斑马鱼中分离细菌菌株，斑马鱼也为使用GF动物的感染筛选的潜在益生菌提供了物种来源（图5）。在该协议中，我们描述了传统的解剖方法来获得肠内容物，而样本也可以直接从活的麻醉鱼中收集（数据未显示）。
<ol><li>选择健康的成年斑马鱼，并用无菌水清洗。在洁净工作台上执行以下步骤。</li>
	<li>用 100 mg/L MS-222 麻醉鱼 5-10 分钟。</li>
	<li>使用 75% 的酒精处理鱼的体表。</li>
	<li>解剖鱼肠并用TSB或BHI培养基挤出肠内容物。</li>
	<li>在有氧和厌氧条件下，通过应用 TSA、血板、TSB 和 BHI 培养基孵育肠道上清液。</li>
	<li>翻译细菌以获得信号菌株并记录不同菌落的特征。</li>
	<li>将细菌储存在-80°C的30%甘油中。</li>
	<li>然后，通过基因组DNA提取和PCR测序鉴定肠道细菌。 注:细菌基因组DNA提取的主要步骤包括液体离心，RNase A和蛋白酶K解离，乙醇提取，冲洗和溶解，按照制造商的说明使用市售试剂盒（参见 材料表）。</li>
	<li>使用美国国家生物技术信息中心 （NCBI） 和基本局部比对搜索工具 （BLAST） 与细菌和古细菌数据库40,41 分析 16S 序列（参见材料表）。</li>
	<li>选择最匹配的细菌和性状，以在属水平上鉴定分离的肠道菌株。</li>
	<li>使用市售试剂盒检测细菌菌株的代谢功能（参见 材料表）。</li>
</ol>8. 流感标签、感染和细菌在GF斑马鱼肠道中的定植
注意:在感染GF斑马鱼之前，用染料修饰分离的细菌并进行计数，使微生物定植和迁移在 体内 连续可见（图6）。
<ol><li>选择宿主中潜在的有益细菌或丰度高的优势菌株来感染GF鱼模型。 注:本研究中使用的细菌是 气单胞菌 属和 弧菌 属（见 材料表），选自实验室中从成年野生型斑马鱼中分离和鉴定的 8 个属42。</li>
	<li>用CM-Dil染料标记新鲜细菌（参见材料表），并将它们暴露于浓度为10 6-108菌落形成单位（CFU）/ mL的5 dpf的GF斑马鱼中。</li>
	<li>在荧光显微镜下用3×放大倍数观察荧光，以指示细菌在6 dpf和14 dpf的GF斑马鱼幼虫肠道中的定植和分布。</li>
	<li>在每个时间点通过平板培养手动计算GF鱼中的细菌数量。</li>
	<li>检测菌落和序列，以确保目标细菌菌株感染成功。</li>
	<li>收集细菌感染的GF鱼样本进行后续检测，包括神经行为、发育指标、组织病理学分析和激素测量等43,44,45。 注意:感染实验中使用的细菌应为新回收的细菌，并通过液体培养基新鲜培养。</li>
</ol>

优化 GF 斑马鱼模型:生成、维护和微生物研究

GF 卤虫 的方案已被中国国家知识产权局授予专利，在获得作者许可后，将解除该方法的使用限制。提交人声明，他们没有其他竞争利益或经济利益。

GF鱼类和GF食品制备协议中的关键步骤 在GF鱼模型的生成过程中，涉及几个关键步骤，包括无菌材料的制备、胚胎的灭菌、GZM的每日更新、各种样品的收集以及使用多种方法对每个样品进行无菌检查。在这些步骤中，胚胎的初始处理对于GF模型的成功是基础和决定性的。控制药剂、药剂浓度和处理时间对于实现最佳灭菌率和孵化成功率至关重要。在将GF鱼胚胎处理到幼虫时，需要特别...

微生物群（即古细菌、细菌、真核菌和病毒）在维持宿主健康方面发挥着至关重要的作用，并通过在肠道屏障、上皮表面和个体粘蛋白功能内的共生相互作用影响生理和病理过程，从而促进各种疾病的发展 1,2,3.从婴儿期到少年期、成年期和衰老等不同生命阶段的微生物群组成，以及其在鼻孔、口腔、皮肤和肠道部位等不同部位的存在，是由不同的栖息地和环境动态塑造的4.生物体中的肠道菌群参与营养吸收、免疫反应、病原体入侵、代谢调节等 5,6.对患者的研究表明，肠道菌群的破坏与人类肥胖、睡眠障碍、抑郁症、炎症性肠病 （IBD）、神经退行性疾病（帕金森氏症、阿尔茨海默氏症）、衰老和各种癌症有关 7,8,9。此外，肠道微生物群和宿主之间的相互作用途径涉及炎症因子、神经递质、代谢物、肠道屏障和氧化应激，正如之前使用小鼠和鱼类模型的研究中观察到的那样10,11。
最近，在临床和动物模型中，已经探索了多种与细菌相关的方法或疗法，包括潜在的益生菌和粪便微生物群移植 （FMT），用于治疗这些疾病。这些探索基于与微生物群-肠-脑/肝/肾轴、微生物群衍生产物和受体活性改变相关的发现12,13。然而，由于微生物群落的复杂性和生成强大的类人疾病模型的挑战，微生物群-宿主系统的发育、各种功能和机制仍未完全理解和识别。
为了解决这些问题，无菌（GF）动物模型在19世纪中叶被紧急提出，并在20世纪主要发展起来。随后的改进，包括抗生素处理和gnotobiotic模型，以及微生物检测和观察技术的进步，进一步完善了这些模型14,15,16。GF动物通过消除自身背景和避免环境微生物而产生，为探索微生物与其宿主之间的相互作用提供了一种极好的策略17。通过应用动物模型和精细的方案，研究人员成功地复制了在GF小鼠和鱼类患者中发现的相似微生物组成。此外，其他GF动物模型，如狗、鸡和猪，作为研究对象提供了多样化的选择18,19,20,21。这种方法使人们能够研究共生微生物组对各种疾病的潜在治疗效果，包括人类癌症免疫疗法16,18。GF模型为特定细菌在宿主内定植、迁移、繁殖和相互作用的特征和机制提供了更准确的见解。这为微生物群相关疾病的发生和发展提供了重要的新见解22,23。GF斑马鱼在微生物研究中的建立和应用的历史从2004年Rawls等人的报告和2006年Bates等人的报告发展到2017年Melancon等人的方案16,24,25。然而，成年或繁殖GF模型的可行性仍然是一个漫长的过程，伴随着可变的寿命、成功率和健康挑战。
在各种动物模型中，斑马鱼（Danio rerio）因其与人体器官和基因组学的有利相似性、发育周期短、繁殖力高和胚胎透明等优点，成为基础和生物医学研究的重要工具19,26。斑马鱼作为可靠的人类疾病模型，提供了体内生理和病理过程的视觉表示，提供了对宿主-微生物相互作用的吸引人特征的见解。值得注意的是，斑马鱼表现出独特的细胞谱系，可以对肠道生理学、微生物动力学、性腺和生殖发育、宿主免疫系统的成熟、行为和新陈代谢进行成像27。斑马鱼胚胎在保护性绒毛膜内发育，直到孵化，在受精后 3 天 （dpf） 成为幼虫。它们在 5 dpf 时积极寻找食物，并在受精后 （mpf） 3 个月左右达到性成熟 28。Rawls 等人 24 报道的首次成功的无菌 （GF） 斑马鱼表明，在卵黄吸收后用高压灭菌饲料喂养的幼虫表现出 8 dpf 的组织坏死和 20 dpf 时的总死亡。这表明了饮食的影响或在涉及长期（>7 dpf）GF鱼的实验中考虑外源性营养供应的重要性29。随后的研究改进了GF鱼的生成方案，采用了在不同鱼类模型中完善的无菌食品和方法16。
然而，对 GF 斑马鱼模型的大多数研究都集中在早期生命阶段，涉及细菌感染在 5 dpf 下持续 24 小时至 48 小时，在实验结束时在 7 dpf 之前收集样本 25,30,31。人们普遍认为，包括人类和斑马鱼在内的生物体中的微生物群在生命开始时就被定植，并在生长和发育过程中形成。该成分在成虫阶段保持稳定，宿主体内微生物群的作用在整个生命过程中都至关重要，尤其是在衰老、神经退行性、代谢相关肥胖和肠道疾病方面3.因此，考虑到鱼类幼虫在早期生命中的免疫和生殖系统不成熟，来自存活时间较长的GF动物的观点可以深入了解微生物在宿主器官发育和功能中的作用机制。虽然斑马鱼肠道中的细菌菌株在以前的研究中已被分离和鉴定，为感染 GF 动物模型以选择益生菌或研究宿主中的细菌功能提供了潜力19,25，但 GF 鱼模型的生成和应用主要局限于早期生命阶段。这种局限性归因于复杂的生产过程、高昂的维护成本以及与食物和免疫力相关的问题，阻碍了旨在调查宿主中微生物群的发育和慢性影响的研究工作。
鱼类的存活率、行为、生长、成熟和整体健康状况，尤其是在无菌 （GF） 模型中，受到摄食方式的显着影响，包括从早期幼虫到幼鱼张口期间的营养摄入和吸收32,33。然而，GF鱼类养殖业面临的挑战之一是缺乏合适的不育日粮，这限制了维持幼鱼生长和存活的营养支持的有效性。考虑到GF鱼的发育防御机制和由于缺乏肠道微生物组而导致的消化能力薄弱，解决这个问题对于恢复GF鱼的生命至关重要。在食物方面，活盐水虾（卤虫属）是张口幼鱼到幼鱼最合适的食物。据观察，与用煮熟的蛋黄或其他天然和合成饵料喂养的鱼相比，用活盐水喂养的鱼表现出更高的生长率和成活率34。虽然GF鱼的早期生活模型可以在卵黄支持下生存，而GF幼虫模型可以通过无菌喂养来维持，但从幼虫到幼鱼并达到性成熟的长期模型仍然具有挑战性。此外，片状或粉状食物受到营养成分不均的限制，并可能影响水质。相比之下，活卤虫具有在盐水和淡水中都能存活、体型小、适合幼虫到成虫、易于配料和更高的孵化质量等优点35.在先前方法16、24、30 的基础上，我们简化了复杂的处理过程，并通过建立易于孵育的 GF 活卤虫作为无菌食品，其持续时间比早期 GF 鱼更长，从而解决了饮食挑战。
本研究提出了一个优化的方案，涵盖 （1） 代、（2） 维持、（3） 确定不育率以及 （4） 维持和饲喂，以确保无菌 （GF） 斑马鱼从胚胎生长到幼虫和幼年阶段。研究结果为GF斑马鱼的孵化、存活、生长和不育提供了初步证据，并为GF 卤虫 属作为不育食品提供了重要指标。无菌活食模型生成和制备的详细步骤为构建和应用长期GF鱼模型以及 GF卤虫 属在微生物群-宿主相互作用研究中提供了重要的技术支持。该协议解决了GF鱼模型上的细菌分离，鉴定和感染，概述了细菌荧光标记的方法，并在显微镜下观察它们在鱼肠道中的定植。GF鱼、细菌感染的gnotobiotic鱼或转移的人类微生物群模型将进行各种检测，以阐明其功能和对宿主免疫、消化、行为、转录组调控和代谢方面的影响。从长远来看，该协议可以扩展到不同的野生型鱼类，例如海洋青鳉鱼，并可能扩展到与特定组织或疾病相关的其他选定的转基因斑马鱼品系。

<table><tbody><tr><td>AB-GZM</td><td>Amphotericin:Solarbio;&amp;nbsp; kanamycin:Solarbio; Ampicillin:Solarbio.</td><td>Amphotericin:CAS:1397-89-3;&lt;br/&gt; kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.</td><td>49.6 mL GZM, 50 &amp;micro;L amphotericin stock solution (250 &amp;micro;g/mL), 25 &amp;micro;L kanamycin stock solution (10 mg/mL), and 250 &amp;micro;L ampicillin stock solution (20 mg/mL).</td></tr><tr><td>1.5 mL, 15 mL, 50 mL EP tubes</td><td>biosharp</td><td>BS-15-M</td><td>To collect samples, and hold agents</td></tr><tr><td>2.4 g/L NaClO</td><td>XILONG SCIENTIFIC Co., Ltd.</td><td>CAS: 7681-52-9</td><td>Diluted with 8% sodium hypochlorite aqueous solution.</td></tr><tr><td>6-well plates, 24-, 48- well plates</td><td>LABSELECT&amp;nbsp;</td><td>11112</td><td>To culture fish</td></tr><tr><td>&lt;em&gt;Aeronomas&lt;/em&gt;</td><td>NCBI database</td><td>No.MK178499</td><td>2019-JPP-ESN</td></tr><tr><td>Anaerobic TSA plates</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;&lt;br/&gt; agar powder:BioFroxx.</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;&lt;br/&gt; agar powder:9002-18-0.</td><td>The TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, and 6 g agar powder under the anaerobic system.</td></tr><tr><td>Anaerobic work station</td><td>GENE SCIENCE</td><td>E200G</td><td>Bacterial isolation, sterile testing</td></tr><tr><td>Analysis</td><td>GraphPad Prism 5</td><td>v6.07</td><td>To analysis the data</td></tr><tr><td>API 20 E kits</td><td>&amp;nbsp;BioMerieux SA, France</td><td>No.1005915090</td><td>Ref 20100 Kits to detect bacterial metabolism</td></tr><tr><td>&lt;em&gt;Artemia &lt;/em&gt;(Brine shrimp)</td><td>Shangjia Aquarium Co., Ltd.</td><td>Aquamaster brand</td><td>Artemia cysts, and brine shrimp eggs&amp;nbsp;</td></tr><tr><td>Auto cycle system for fish culture</td><td>Ningbo Hairui Technology Co., Ltd</td><td>No Cat</td><td>Maintain the fish</td></tr><tr><td>Autoclave</td><td>Zeal Way</td><td>G154DWS</td><td>Prepare the materials</td></tr><tr><td>BHI Aerobic</td><td>Coolaber</td><td>Cat#PM0640</td><td>BHI medium was prepared, wherein 100 mL medium included 3.7 g BHI powder.</td></tr><tr><td>BHI Anaerobic</td><td>Coolaber</td><td>Cat#PM0640</td><td>BHI medium was prepared and divided into anaerobic tubes under the anaerobic system.</td></tr><tr><td>Biochemical incubator</td><td>LongYue Co., Ltd</td><td>SPX</td><td>For fish and plates</td></tr><tr><td>Biosafety cabinet</td><td>Haier</td><td>HR40-IIA2</td><td>Sterile treatment and testing</td></tr><tr><td>Bleaching agent of 0.02 g/L NaClO</td><td>XILONG SCIENTIFIC Co., Ltd.</td><td>CAS: 7681-52-9</td><td>Working solution with sodium hypochlorite (NaClO) concentration: Diluted with 8% sodium hypochlorite aqueous solution or 166.6 uL 6% sodium hypochlorite with 500 mL distilled water.</td></tr><tr><td>Blood plates</td><td>sheep blood:Solarbio</td><td>Cat. NO. TX0030</td><td>Sterile-defibrinated sheep blood was added into TSA to prepare 5% blood plates.</td></tr><tr><td>Cell culture flask</td><td>Corning</td><td>430639</td><td>To culture fish</td></tr><tr><td>CM-Dil dyes</td><td>Molecular Probes</td><td>Cat#C7000&amp;nbsp;&amp;nbsp;</td><td>To label the bacteria</td></tr><tr><td>Constant temperature shaking incubator</td><td>Peiving Co., Ltd</td><td>HZQ-X100</td><td>Bacterial culture</td></tr><tr><td>Database</td><td>NCBI</td><td>Bacteria and Archaea database</td><td>Link: Archaea FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Archaea/&lt;br/&gt; Bacteria FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Bacteria/</td></tr><tr><td>Disposable Pasteur pipette</td><td>biosharp</td><td>bs-xh-03l</td><td>Used to change water, and transfer eggs</td></tr><tr><td>Disposable petri dish</td><td>biosharp</td><td>BS-90-D</td><td>To culture fish</td></tr><tr><td>DNA kits</td><td>Solaribio</td><td>Cat#D1600</td><td>Bacterial genomic DNA extraction kits&amp;nbsp;</td></tr><tr><td>Electric pipette</td><td>SCILOGEX</td><td>Levo me</td><td>Change water</td></tr><tr><td>Exiguobacterium</td><td>NCBI database</td><td>No.MK178504</td><td>2019-JPP-ESN</td></tr><tr><td>GZM</td><td>Sea salt:LANDEBAO Co., Ltd.</td><td>No Cat</td><td>Composed of 1 L of water and 1.5 mL of sea salt solution (40 g/L), autoclaved. The content of sea salt in the GZM solution was 60 mg/L.</td></tr><tr><td>Laboratory pure water system</td><td>Hitech Co., Ltd</td><td>Prima-S15</td><td>Prepare the agents</td></tr><tr><td>Microscope</td><td>Nikon</td><td>SMZ18</td><td>With fluorescent light to observe fish larvae</td></tr><tr><td>PCR kits</td><td>TIANGEN</td><td>Cat#ET101</td><td>Taq DNA Polymerase kit</td></tr><tr><td>Pipette</td><td>LABSELECT&amp;nbsp;</td><td>sp-013-10</td><td>Change water</td></tr><tr><td>Povidone iodine (PVP-I)</td><td>Aladdin</td><td>Lot#H1217005</td><td>Aqueous solution povidone iodine 0.4 g/L pure water.</td></tr><tr><td>Timing converter</td><td>PinYi Co., Ltd</td><td>AL-06</td><td>To regulate the light</td></tr><tr><td>TSA plates</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;&lt;br/&gt; agar powder:BioFroxx.</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;&lt;br/&gt; agar powder:9002-18-0.</td><td>TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, 6 g agar powder.</td></tr><tr><td>TSB Aerobic</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;</td><td>TSB medium was prepared, wherein 400 mL medium included 6 g tryptone, 2 g soy peptone, and 2 g NaCl.</td></tr><tr><td>TSB Anaerobic</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;</td><td>TSB medium was prepared and divided into the anaerobic tubes under the anaerobic system.</td></tr><tr><td>Ultra-clean workbench</td><td>Airtech</td><td>SW-CJ-2FD</td><td>Sterile treatment and testing</td></tr><tr><td>Ultra-pure flow system for fish culture</td><td>Marine Biological Equipment company</td><td>No Cat</td><td>Produce water for fish</td></tr><tr><td>&lt;em&gt;Vibrio&lt;/em&gt;</td><td>NCBI database</td><td>No.MK178501</td><td>2019-JPP-ESN</td></tr></tbody></table>

generation, maintenance, and identification of germ-free zebrafish models from larvae to juvenile stages

斑马鱼因其与哺乳动物的基因组相似性、在相对干净的绒毛膜环境中发育的透明胚胎以及与啮齿动物模型相比幼虫发育极快，因此可以作为生长、免疫和肠道微生物群研究的宝贵模型。无菌（GF）斑马鱼（Danio rerio）对于评估污染物毒性和建立与微生物功能相关的类人疾病模型至关重要。与传统饲养（CR）模型（共同饲养的鱼类）相比，GF斑马鱼可以更准确地操纵宿主微生物群，有助于确定微生物与宿主之间的因果关系。因此，它们在促进我们对这些关系的理解方面发挥着关键作用。然而，由于免疫功能和营养吸收的限制，GF斑马鱼模型通常是在生命早期（从胚胎到幼虫）生成和研究的。本研究优化了早期GF斑马鱼模型的生成、维持和鉴定，无需摄食和长期使用GF食物（如 卤虫 、盐水虾）摄食。在整个过程中，每天进行采样和培养，并通过多次检测进行鉴定，包括平板和 16S rRNA 测序。记录GF斑马鱼的无菌率、存活率和发育指标，保证所生成模型的质量和数量。重要的是，这项研究提供了关于GF鱼的细菌分离和感染技术的详细信息，使得在GF食物支持下能够有效地创建从幼虫到幼鱼阶段的GF鱼模型。通过在生物医学研究中应用这些程序，科学家们可以更好地了解肠道细菌功能与宿主健康之间的关系。

GF斑马鱼模型可以通过利用成对的斑马鱼产卵来有效地生产，并根据以前的GF鱼模型35优化了协议。单个 6 孔板可培养约 30-48 个胚胎/幼虫，可进行充足的数据收集和统计分析。无菌处理后，将GF胚胎在干净的培养箱中培养至48-72小时孵化为幼虫，并每天检测收集的样品以改变GZM，这对于保持无菌状态至关重要（图1）。7 dpf后，应为幼虫至幼虫阶段准备蛋黄和?...

Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

作者聚焦:建立无菌斑马鱼用于微生物群-宿主关系研究

该协议概述了获得无菌（GF）鱼胚胎并将其从幼虫维持到幼虫阶段的主要步骤，包括取样和检测其不育状态。使用GF模型处理感染对于理解微生物在宿主健康中的作用非常重要。

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

这项研究的范围是生成新颖的、无菌的动物模型，这将是研究各种微生物组功能的有用和强大的工具。在该领域，早期的无菌鱼模型是最近才得到应用的，但由于养殖条件和工作人员发育防御的不同，长期模型仍然有限。目前的GF鱼模型通常是早期生命的。因此，包括无菌秤、样品检测方法和提供无菌食品在内的技术是需要克服的非常重要的挑战。无菌斑马鱼模型对于研究宿主-微生物群相互作用及其对发育、免疫和疾病的影响至关重要。该模型使研究人员能够精确地操纵单个肠道细菌，并观察其对透明斑马鱼胚胎的影响。它为遗传疾病的肠道微生物的作用提供了宝贵的见解。未来，我们的研究将主要集中在建立斑马鱼全生命周期的无菌模型上，以更好地了解肠道微生物组的功能。

generation-maintenance-identification-germ-free-zebrafish-models-from

Research

JoVE Journal

Developmental Biology

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

1.0K 次观看.  Chongqing Medical University. 这项研究的范围是生成新颖的、无菌的动物模型，这将是研究各种微生物组功能的有用和强大的工具。在该领域，早期的无菌鱼模型是最近才得到应用的，但由于养殖条件和工作人员发育防御的不同，长期模型仍然有限。目前的GF鱼模型通常是早期生命的。因此，包括无菌秤、样品检测方法和提供无菌食品在内的技术是需要克服的非常重要的挑战。无菌斑马鱼模型对于?...

视频: 作者聚焦:建立无菌斑马鱼用于微生物群-宿主关系研究

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

科研

遗传学

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

生物学

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 ...

癌症研究

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

发育生物学

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

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 ...

An Introduction to the Zebrafish: Danio rerio

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their high degree of genetic conservation with humans and their simple, inexpensive maintenance. Additionally, gene expression can be easily manipulated in zebrafish embryos, and their transparency allows for observation of developmental processes.
This overview video first introduces basic zebrafish biology, including their phylogeny, life cycle, and natural environment, before presenting the features that make them so useful in the lab. A brief history of zebrafish research is also provided through a review of major discoveries made in fish, ranging from the early establishment of methods for efficient genetic screening to the discovery of novel therapeutics for human diseases such as cancer. Finally, some of the many avenues of experimentation performed in zebrafish are discussed, including immunological and developmental studies.

Danio rerio Zebrafish as a Model Organism

印度斑马鱼:斑马鱼的介绍

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their ...

教育

JoVE Science Education

基础生物学

模式生物II:小鼠，斑马鱼和鸡

Zebrafish Maintenance and Husbandry

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to their small size, large numbers of zebrafish can be housed in the laboratory at low cost. Although zebrafish are relatively easy to maintain, special consideration must be given to both diet and water quality to in order to optimize fish health and reproductive success.
This video will provide an overview of zebrafish husbandry and maintenance in the lab. After a brief review of the natural zebrafish habitat, techniques essential to recreating this environment in the lab will be discussed, including key elements of fish facility water recirculation systems and the preparation of brine shrimp as part of the zebrafish diet. Additionally, the presentation will include information on how specific zebrafish strains are tracked in a laboratory setting, with specific reference to the collection of tail fin samples for DNA extraction and genotyping. Finally, experimental modifications of the zebrafish environment will be discussed as a means to further our understanding of these fish, and in turn, ourselves.

Maintenance and Husbandry of Danio rerio Zebrafish

斑马鱼的养护和饲养

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to ...

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

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