Name: the complete and updated "rotifer polyculture method" for rearing first feeding zebrafish
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在保健和本协议描述的代表性结果产生鱼的使用是完全按照规定的机构动物护理和使用委员会在波士顿儿童医院，协议＃14-05-2673R的指导方针进行。

1.轮虫文化<ol><li> 文化系统使用的是100升培养容器基本组件 <ol><li>收集所有组件的轮虫培养设置。轮虫培养装置由一个培养容器（CV）发展壮大的轮虫;类似的船只，以保持喂料轮虫（喂料培养容器，FCV）;圆底孵化缸（订阅水库，FR），用于存储所述藻类饲料混合物（AFM）的;气源（AS），以充气的简历，燃料电池汽车和FR;蠕动泵计量定时器（PMT）来控制藻类交付饲料进入CV和燃料电池汽车;和牙线微粒过滤器（FPF），坐落在简历里面。 注 :耗材和组件的完整列表中的材料清单提供。 </li></ol></li><li> 组态 <ol><li>提升支架上或桌子上的简历和燃料电池汽车，这样的文化可以通过排水管装修成一个集合续轻松收获ainer（ 图1）。使用灵活的送风管到AS连接到每个培养容器的长度刚性管。确保管足够长以将空气输送到CV或燃料电池汽车的底部。 </li><li>使用小容量的空气线到AS连接到一个长度刚性管的延伸到包含AFM对FR的底部。安装一个阀门到每个空气管路调节风量。帧中继连接到与饲料输送管的PMT和运行从PMT的管导入钻入CV / FCV的一侧，在顶部附近的孔。 图1。 </li></ol></li><li> 启动 <ol><li>培养容器填充到90％的可用容量与反渗透水（RO）的。如果RO不可用，使用清洁，脱氯市政供水;然而，应该执行一个生物安全风险评估，以确保没有潜在的致病微生物存在于水源。注:这种分析，可以执行任何资质的水质检测实验室。 </li><li>剂量的培养容器水与水族箱盐达到15克/升的盐度。置的空气流进入容器，使得它保持一个"滚动沸腾"，然后慢慢盐的测定量添加到培养容器，直到它完全被曝气溶解。继续曝气水为&gt; 1小时，以确保其完全氧化。 </li><li>使藻类进料混合物。向3升的干净，脱氯鲜（0ppm的）水加入100克碳酸氢钠和100克的氨中和剂（钠hydroxymethylsulfonate）。这最后的试剂提供中和从自来水或漂白残基的任何残余的氯从消毒的培养设备的额外的好处。以确保这些化合物完全溶解，这一点至关重要。然后加入1升藻浓缩液（生物干重〜15％）。添加进料混合物，以FR和储存在4℃。 </li><li>加5的首发文化年 - 1000万元<eM&gt;含充气15克/ L矿化度水褶皱臂尾轮虫轮虫的简历。如果轮虫已装运或储存过程中被冷却，它们应该是逐渐（在30分钟期间或更长的时间）驯化的水在培养容器中的温度（25 - 27℃）。 </li><li>打开PMT，并开始抽藻送入轮虫培养容器。使用PMT的定时器功能，设置藻类饲料混合物的排出流量，使得〜1.6 ml的藻类饲料混合物的每百万轮虫在培养递送，每天。分布在一小部分的喂食定期超过24小时的周期过程;越频繁喂食，效果更佳。 </li><li>通过手动开启PMT为在设定时间内（例如，1分钟）校 ​​准泵的输出率，并收集它此间隔成量筒或烧杯中泵藻类。例如，如果该PMT剂量5 ml的藻类中1分钟，然后将剂量率将是5 ml的藻类/分钟。 </li><li>乘以本轮虫的数以百万，1.6 ml的计算所需的日常饲养率。例如，对于在100万轮虫人口尺寸的轮虫培养需要〜160 ml的饲料，每天（100×1.6毫升）的。 </li><li>设置的PMT，在整个24小时内的剂量以规则间隔的总日饲料需求。例如，递送的160毫升的总日饲料量可以以能够部分被递送一次，每3小时，在24小时内使用的PMT组为5毫升/分钟4分钟，每日8次的计量泵速率（5毫升/分钟×4分钟= 20毫升×8喂食= 160毫升）。 </li><li>让文化成长，直到它生成所需的人口，一般为48 - 72小时，收获前。在24小时后启动，添加牙线粒子滤波器到培养容器中，并开始正常的维护。 </li></ol></li><li> 保养 注:培养运行在一个连续的基础上，并要求routin而理想地应在同一时间的每一天进行，按照以下顺序ë维修。 <ol><li>燃料电池汽车填充到90％的有效量的清洁，脱氯淡水，给药10克/升的水族馆的盐。确保水充分混合，并且所有的盐的完全溶解。置的空气流进入容器，使得它保持一个"滚动沸腾"。测量盐度用折射并确保盐度为10克/升。要达到这个目标，不要超过它，这一点至关重要。 </li><li>样品中的CV轮虫:确保培养充分混合，然后收集3个样本的2 - 3各自使用一个移液管或autopipettor毫升，从培养的不同部分。结合这些样品中的管或小瓶方便的大小（例如，10ml）中的。 </li><li>转移1 - 2毫升组合样本的上一个培养皿，以便它可以在解剖显微镜下观察。检查文化素质（游泳的行为轮虫，存在脱落的鸡蛋，污染原生动物）。 </li><li>通过加入100微升50％Lugols碘溶液到样品固定剩余组合的样品中的轮虫。在几秒钟内加入Lugols后，观察轮虫停止游泳。现在，很容易算轮虫。 注:乙醇，稀漂白剂，或醋可以代替Lugols的使用。醋（2滴/ 10毫升）中有被优点无危险，没有失去强度中存储作为漂白和碘溶液可以，并没有使轮虫合同，所以纤毛的电晕和"脚"保持伸出并且动物看起来更自然。 </li><li>确保样品充分混合（固定轮虫会沉淀迅速），然后迅速采取约2ml的子样本在一个塑料吸管和分配1 ml的成塞奇威克-拉夫特计数滑动件（20×50 1毫米正方形）（图2 ）。使用解剖或复合式显微镜，算上完整轮虫和Total号码附着到这些轮虫蛋（图2）。计数一样多的滑动面积的必要来算〜100轮虫。计算每毫升轮虫的数量，并在电子表格或日志记录此。 </li><li>收获〜于CV轮虫的体积的30％:除去空气供应和牙线过滤，慢慢在CV的底部打开阀并允许水流入一个浮游生物集电极与一个53微米网状筛。收集过滤成桶或漏极后流出收集器的底部的水。使用温和到中等流量，以免损坏轮虫。不要让轮虫干在屏幕上。 </li><li>出于一致性的原因，建议建立与燃料电池汽车的每一天，标准号轮虫。因此，基于已知的CV轮虫密度和收获量，调整燃料电池汽车总体积达到一致的最终密度（例如，1500轮虫/毫升）。添加收获烤肉FERS到FCV:（ - 24 g / L的10），轻轻地从采集窗口使用的是洗瓶子装满干净的盐水转移轮虫。反转屏幕在燃料电池汽车，并用食盐水的温柔流洗轮虫到燃料电池汽车。启动PMT提供饲料（〜每天每百万轮虫1.57毫升）的燃料电池汽车。 </li><li>磨砂CV的整个内部用干净，柔软的尼龙刷或擦洗垫。 </li><li>使加入盐的适量的清洁RO水测定量在5加仑桶取代的水的体积丢失收获15克/升的水一新的组合。的盐添加到水在水桶和剧烈搅拌，直至其完全溶解，然后添加到CV。 </li><li>在水槽采用高压喷淋，冲洗绒毛过滤器，直到它是免费的碎片，然后将其返回到CV。 </li><li>调整藻类通过改变每个给药事件的持续时间递送到CV的进料速率，根据轮虫/毫升每日计数。使用在步骤1.3.8提供的计算，上述确定进料待递送的适当的量。 </li><li>约24小时后，重复上述过程。开始通过收获残留在燃料电池汽车（即没有所需的前一天）的轮虫中相同的方式，如上所述（步骤1.4.2 - 10年1月4日）。集中起来在2升的新鲜，洁净的脱氯水（5克/升的盐）。这些可以存储在4℃下作为备用电源，或用于饲料的鱼后期阶段，超出了在该协议描述。 注意:此协议允许高达2 - 3天减压培养维护（与正常的自动喂养），因为在CV轮虫可以容忍收成遗漏无严重的后果。 </li></ol></li></ol> 2.混养<ol><li> 建立 <ol><li>通过茶型过滤器浇催生的胚胎，然后用无菌FIS轻轻漂洗收集从产卵事件斑马鱼胚胎h进水（或适当地调节溶液的其它任何未污染源;例如，胚胎培养基，E3等）从一个洗涤瓶入培养皿中。 </li><li>孵育胚胎在25 - 28℃下在培养皿中在40密度 - 每皿50胚胎5天。 </li><li>开始第5天受精后，或当大于90％的孵幼虫的正在积极游动在水柱混养相。 </li></ol></li><li> 接种 <ol><li>添加500毫升轮虫培养直接从FCV到3.5升育苗池;列入轮虫养殖水体藻类提供饲料用于维护轮虫混养过程中的营养成分。 </li><li>从一个培养皿中的幼虫轻轻倒入幼儿园坦克。确保没有幼虫留在菜。 </li><li>从再循环系统或专用水源加入500毫升干净，空调鱼水到容器达到1升的终体积和最终的小号alinity 5克/升。 注意:因为如果盐度是&gt; 7克/升和轮虫生存将受到负面影响，斑马鱼幼体生存将受到负面影响这盐度是至关重要的，如果盐度&lt;2克/升。 </li></ol></li><li> 混养阶段 注:混养相应持续长达4天接种后（共5天，对应于天5-9受精后）。 <ol><li>在此期间观察混养罐至少每天一次，以确保轮虫和鱼存在和生长。确保轮虫在整个水柱可见。确保了fishs也是可见的水柱内，轮虫中游泳。 </li><li>开始正常的水流量通过罐。放置一个屏幕或挡板在泄油口，以确保幼虫不会被清理出的坦克。 注:在此阶段结束时，鱼会大到足以消耗较大的猎物如卤虫125微米 - 在75的尺寸范围内的无节幼体或饲料加工物品。 注意:一代表混养罐内的轮虫种群动态通过取样测定/如步骤1.4.2描述计数从以相同的方式将罐轮虫 - 1.4.5。这是从混养阶段开始，直到完成完成每天一次。 </li></ol></li></ol>

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EC亨利是由芦苇海水养殖公司，该公司提供的轮虫，藻类的浓缩，和其他物资，以水产养殖和鱼类爱好者市场就业。

成功实施轮虫混养方法喂养早期幼虫的斑马鱼，需要有效的协议两个任务:第一喂养以及在同一水箱轮虫斑马鱼幼体连续轮虫培养体系的建立和维护喂鱼，和培养。 设置用于连续盐水轮虫生产系统首先由劳伦斯和共同作者14所述斑马鱼的实验室已被修改和增强的多种方法，使得它更加在应用健壮和普遍。新的协议包含改进都在设备和方法。 存储系?...

斑马鱼（ 斑马鱼 ）是一个卓越的实验动物在越来越多的科学学科的利用，包括但不限于发育遗传学，毒理学，行为，水产养殖，再生生物学，以及许多人类疾病1的造型- 5。虽然品种相对容易在实验室中保持，有许多与它们培养6相关联的管理的挑战。最突出的是苗种培育，特别是当鱼第一次开始喂气膀胱充气7其后。在正常，控制的条件下，此发育事件发生在〜5天后受精（DPF），但有以下3 -生长5天是特别关键的7。在这一阶段的中心技术难度是充分满足第一馈送幼虫的营养需求 - 饲料项目必须适当尺寸，diges1149，有吸引力，并在几乎连续的基础上提供，而不在培养罐产生过多的浪费。历史上，这已经达到一般通过提供大量的少量饲料鱼的坦克，以及常规的水交换8,9。虽然这些方法有一定程度的成功，它们是效率低下的，要求高劳动投入，并返回唯一的变量和生长和存活10的速率限制。 在自然界中，斑马鱼幼体想必饲料丰富的小礼物在水体中浮游动物11。出于这个原因，结合了活饲料如草履虫 ，轮虫和卤虫 larviculture协议通常是最有效的7。 2010年，百思及其合作者表明，它是可能的静态，苦咸水以及海水轮虫的第5天的外源性饲养12幼虫生长的斑马鱼。这种方法，它利用ES轮虫培养的天然高生产率，以提供充足的，高营养的猎物，不污染水，幼虫生长和存活的低劳动力投入12,13的收益率非常高的速度。近年来，越来越多的世界各地的实验室都采用这个协议的变化，现在很多都培养轮虫以连续的方式来支持育苗系统14。 在过去的几年中，为轮虫/斑马鱼混养和轮虫的生产方式进行了细化和改进，变得更加标准化和轻松扩展。本文提供了一步一步的说明1）连续和强大的轮虫生产及2）建立用于支持鱼类生长健壮的第5天的外源性摄食轮虫/斑马鱼混养系统。

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Rotifer Culture Infrastructure&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>100 L Culture Vessel</td><td>Aquaneering</td><td>Custom</td><td>Polycarbonate culture vessel, conical bottomed, with drain valve</td></tr><tr><td>5 Gallon Culture Bucket Kit</td><td>Reed Mariculture</td><td>CCS Starter Kit</td><td>Small volume culture vessel for small facilities</td></tr><tr><td>Rigid Clear Tubing 1/2&quot; O.D., 36&amp;rdquo;</td><td>Pentair Aquatic Ecosystems</td><td>16025</td><td>Rigid clear tubing for air delivery</td></tr><tr><td>Mesh tube</td><td>Pentair Aquatic Ecosystems</td><td>RT444X</td><td>Mesh tube support for floss filter</td></tr><tr><td>Rotifer Floss</td><td>Reed Mariculture</td><td>Rotifer floss 12&amp;rdquo; x 42&amp;rdquo;</td><td>Particulate waste trap</td></tr><tr><td>Peristaltic Metering Timer Pump, 5 GPD</td><td>Grainger</td><td>38M003&amp;nbsp;</td><td>Metering pump with timer for dosing feed to rotifers</td></tr><tr><td>Peristaltic Metering Timer Pump, 1-100 mL/hr (for smaller-scale culture)</td><td>Coral Vue</td><td>SKU: IC-LQD-DSR</td><td>Metering pump with timer for dosing feed to rotifers</td></tr><tr><td>Silicone Tubing&amp;nbsp;</td><td>Cole Parmer</td><td></td><td>Tubing for algae delivery to rotifer vessel</td></tr><tr><td>Rigid Clear Tubing &quot; O.D.,36&amp;rdquo;</td><td>Pentair Aquatic Ecosystems</td><td>16025</td><td>Rigid clear tubing for air delivery to algae paste</td></tr><tr><td>Rigid Clear Tubing O.D., 36&amp;rdquo;</td><td>Pentair Aquatic Ecosystems</td><td>16025</td><td>Rigid clear tubing for algae delivery</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Rotifers&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Live Rotifers Brachionus plicatilis Type L</td><td>Reed Mariculture</td><td>Type L 5 million</td><td>Rotifer stock culture for system startup</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Rotifer Feed&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium hydroxymethylsulfonate</td><td>Reed Mariculture</td><td>ClorAm-X&lt;sup&gt;&amp;reg;&lt;/sup&gt; 1lb tub</td><td>Ammonia reducer for algae feed mix</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific</td><td>S25533B</td><td>pH buffer for algae feed mix</td></tr><tr><td>Microalgae concentrate</td><td>Reed Mariculture</td><td>Rotigrow Plus&lt;sup&gt;&amp;reg;&lt;/sup&gt; 1 liter bag</td><td>Nutritionally optimized rotifer feed</td></tr><tr><td>RG Complete</td><td>Reed Mariculture</td><td>RG Complete 6 oz bottle</td><td>All in one microalgae based feed for small scale cultures</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Water Preparation&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Reef Crystals Reef Salt</td><td>That Fish Place</td><td>198210</td><td>Salt for making culture water (NOTE: this item is an example only; any contaminant free salt formulations may be used).&amp;nbsp;</td></tr><tr><td>Refractometer</td><td>Pentair Aquatic Ecosystems</td><td>SR6</td><td>measuring salinity</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Rotifer Culture Equipment&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Plankton Collectors 12&quot; Dia, 53 microns</td><td>Pentair Aquatic Ecosystems</td><td>BBPC20</td><td>Mesh screen for collecting rotifers</td></tr><tr><td>Scrub Pads</td><td>Pentair Aquatic Ecosystems</td><td>SCR-58</td><td>Scrub pad for cleaning inside of culturing vessels</td></tr><tr><td>Scrub Brush</td><td></td><td></td><td></td></tr><tr><td>Bucket</td><td>Grainger Supply&amp;nbsp;</td><td>43Y530</td><td>Graduated bucket for mixing culture water</td></tr><tr><td>Hatching Jar</td><td>Pentair Aquatic Ecosystems</td><td>J30</td><td>Storage of algae feed mix</td></tr><tr><td>Lugol&amp;rsquo;s Solution, Dilute</td><td>Fisher Scientific</td><td>S99481</td><td>Agent used to immobilize live rotifers for counting</td></tr><tr><td>Sedgewick-Rafter plankton counting slide with grid&amp;nbsp;</td><td>Pentair Aquatic Eco-Systems</td><td>M415</td><td>Counting rotifers</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Miscelleneous&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Tea Strainer</td><td>Kitchenworks</td><td>971972</td><td>Used for collecting zebrafish embryos after spawning</td></tr></tbody></table>

the complete and updated "rotifer polyculture method" for rearing first feeding zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

这里所描述的连续轮虫培养系统是动态的，这是正常的轮虫数量的波动在小范围内随着时间的推移，如果有变化，日常饲养和收获率。在水产养殖设施在波士顿儿童医院的活性培养物中的一个的轮虫的人口，保持在上述的方式，进行30 天 （图3）监控。在此期间，平均培养密度为932轮虫/毫升，最大为1330轮虫/ ml和至少510轮虫/毫升。这代表保持按照...

Watch this Scientific Journal Video about The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish at JoVE.com

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

the-complete-updated-rotifer-polyculture-method-for-rearing-first

Research

JoVE Journal

Developmental Biology

15.3K 次观看.  Boston Children's Hospital. Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

视频: The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

科研

遗传学

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

发育生物学

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

优化 GF 斑马鱼模型:生成、维护和微生物研究

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

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

作者聚焦:建立无菌斑马鱼用于微生物群-宿主关系研究

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

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 ...

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 ...

生物学

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 ...

Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

一个经济实惠，易于构建斑马鱼设施建设

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

摘要

探索更多视频

此视频中的章节

使用斑马鱼作为体内模型系统分析 DNA 复制定时

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生

斑马鱼原代细胞的培养与转染

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

定期保养和维护的斑马鱼（

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

一个经济实惠，易于构建斑马鱼设施建设

利用斑马鱼对肠道转运时间进行高通量测量

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

摘要

探索更多视频

此视频中的章节

使用斑马鱼作为体内模型系统分析 DNA 复制定时

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

斑马鱼原代细胞的培养与转染

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

定期保养和维护的斑马鱼（

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

一个经济实惠，易于构建斑马鱼设施建设

利用斑马鱼对肠道转运时间进行高通量测量

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生