这项工作是由以下资金为RAW的支持：美国国立卫生研究院资助K01 DK083512，DP2 OD008470，和R01 DK100237;三月角钱罗勒奥康纳入门学者奖＃5-FY12-75;开始从科学和生物科学系的圣母院书院的大学基金;和丰盛的礼物圣母院大学从伊丽莎白和迈克尔·加拉格尔代表格尔家族培养干细胞研究。资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有任何作用。我们感谢生物科学部的工作人员的支持，和中心斑马鱼研究在巴黎圣母院为他们在我们的斑马鱼殖民地的照顾和福利的杰出贡献。我们感谢GF格拉赫采取在图4中提供的照片。最后，我们感谢我们的研究实验室的所有成员的意见，讨论和INSIG关于这项工作HTS。

注：该程序在本协议中所述的斑马鱼胚胎的工作被批准的机构动物护理和使用委员会在Notre Dame大学。注意：虽然下面的协议提供了一个示范的精子分离，涉及到男性的安乐死，精子可以从生殖孔收集麻醉活斑马鱼的男性使用温和的腹压和微毛细管精子采集17,18。活的男性压缩需要几个男性以用于收集精子可以从一个实施安乐死雄性17,18得到的等效量。但是，已经经过挤压的男性仍相当健康和可用于天然交配或随后在体外程序17。只要有可能，选择的过程中应尽量减少不必要的鱼牺牲。 1，解决方案和斑马鱼交配钱伯斯的制备 &lt;醇&gt; <li>准备汉克的股票解决方案（＃1，2，4，6）和汉克的预混料解决方案（见材料表）19，然后在4℃下高压灭菌器和存储</li><li>选择成年雌性斑马鱼（s）的单倍体离合器收集所需的号码。注：根据经验，采用诱变F1由蒂宾根近交系品系斑马鱼，性成熟的雌性约50％的平均&#39;挤压&#39;将它们放在一个笼交配过夜男性吸他们之后- 即获得了离合器通过挤压过程 - 和大多数的这些离合器（在70-90％之间不等）的​​生产上可行的单倍体。有两个研究人员进行单倍体准备，约80成年雌性斑马鱼的F1可以挤在一个早晨。计算在F1代的两个挤压速率和研究者的可用于执行EXP数量所需的单倍体实验中，因子的试剂eriment。请记住，女性，将生产高品质的单倍体离合器的数量是极其多变，不同品系，年龄和食品饮食15的斑马鱼女性之间观察到的差异。 </li><li>通过将每个成年斑马鱼雌性的成年雄性斑马鱼在水系统的一个腔室，使它们通过一个分割器分离过夜准备交配笼中。注意：女性必须暴露在男性隔夜黄金，或者他们做好准备，通过挤压产卵。 </li></ol>睾丸2。解剖<ol><li>使Hank氏储备溶液＃6新鲜加入0.35克碳酸氢钠至10毫升蒸馏水。拌匀溶解的碳酸氢钠粉末。 </li><li>结合下面的在冰上的管中，以使缓冲Hank氏的精子最终工作溶液：9.9毫升汉克斯预混我的用100μl原液＃6。 </li><li>拌匀，再分装500微升的汉克7，S最后工作溶液倒入离心管置于冰上。注意：每个500μl的等分试样将用于从4-5雄性斑马鱼睾丸制备的精子。 </li><li> 4-5雄性斑马鱼为每个精子收获之间进行选择。注意：这将收获足够的精子进行收集，处理，并存储在一个1.5 ml离心管，它可以被用来施肥约15单倍体离合器。以制备另外的管取决于实验的规模选择的男性额外队列。如果选定的雄性斑马鱼是小型或睾丸切除不完全，通常为5的男性将是必要的。 </li><li>通过使用网对鱼轻轻转移到含0.2％三卡因，在室温下约5-6分钟烹调安乐死每个男性。注意：小心，以确保男性已经开始正常解剖之前安乐死。通常情况下，等待数（2-3）最小的鱼停止移动鳃和鱼后不再响应触摸。 </li><li>轻轻一挑的男性与一个塑料勺子，小心吸干液体的男性干。接着，取出男性用锋利的剪刀头，然后切沿腹正中线切开。注：去除鱼的所有多余的液体是必不可少的，以防止触发激活精子。 </li><li>取下用细镊子和地方变成为出售生物危害容器腹腔肠道和肠相关的器官。 </li><li>用细镊子取出睾丸，分别位于沿背体壁，横向到鱼鳔，并有一个白色的不透明的外观立体显微镜下观察时。注：保持睾丸完好可以帮助防止精子暴露于水性体液，从而防止过早激活。 </li><li>将睾丸成冰的精解，并保持管上的冰与其他睾丸解剖。重复步骤2.6-2.9，直到所有的米麦啤酒已被解剖。 </li><li>将尸体和所有其余的动物组织中转化为适当的机构处理生物危害容器。 </li></ol> 3，紫外线灭活精子<ol><li>轻轻研磨的离心管中，用无菌微管杵均质睾丸。 </li><li>将上清转移到一个小培养皿或冰上表玻璃。注意：不要随上清液转移组织碎片。这将创建阴影和损害精子的紫外线灭活。 </li><li>冲洗精子管300-400之间微升额外汉克的最后工作溶液（步骤2.2并冰冻保存），并添加此上清液洗的小培养皿或手表玻璃在步骤3.2。 </li><li>暴露的盘从一个工作台灯（254纳米），以紫外线为总共2分钟，在从约15（38.1公分）的灯源的距离。注意：用紫外灯工作时一定要小心，并要戴上面罩或另一只眼睛的个人防护装备。 </li><li>返回菜冰桶，然后紫外线灭活的精子转移到一个新的离心管中，样品储存在冰。注意：此时将有大约800微升紫外线灭活的精子为单倍体离合器施肥。精子应保持在冰上整个时间的整个持续时间花在压榨女性。精子在冷汉克的可使用长达6小时无受精结果有任何减少。有趣的是，其他研究人员已经报道，精子在冷Hank氏是可行的从几个小时到几天18。 </li></ol> 4，从成年斑马鱼女和离合器的体外受精鸡蛋采购<ol><li>通过结合将20ml 0.2％三卡因股票80毫升鱼水系统准备洗澡三卡因麻醉。 </li><li>选择女性卵子的收集，使用网络来轻轻地放在她的TR的菜icaine。 </li><li>等待2-3分钟，直至雌性被麻醉并不再响应触摸。注：如果女性抽搐或引发其他的变动，给她更多的时间来屈服于麻醉。 </li><li>用塑料勺子轻轻抬起女性，把女性到纸巾吸走多余的液体倒出之前的解决方案。注意：小心不要让多余的解决方案上的女性，因为液体会增加精子的前触发卵子激活。 </li><li>将女性对她的幻灯片在一个干净的培养皿和可视化的体视显微镜下，她的肚子。 </li><li>中风女性的腹部轻轻地对使用一个或两个手指从一只手从她的腹部挤卵，从另一方面仅次于她的背体壁安放一个或两个手指同时支持女性的背部大约10-20秒。注：在抚摸女性的腹部，鸡蛋应该出来很容易。如果鸡蛋不出现机智ħ缓解它是不可取的“努力努力”，并从女性的力量鸡蛋。这表明，鸡蛋不存在和/或不准备受精，并继续推动与危害女性（ 例如 ，放气鳔或引起其他致命内伤）的相当大的风险。如果女性被挤压，但提供没有或几个鸡蛋，返回到雌性动物设施。 1-2周的休息后，配对与男性的自然交配清理残留的蛋碎片的女性。分离的女性，自然交配成罐的另一个1-2周的休息尝试第二挤压之前。可替换地，女性可以静置4周，在此期间，它们可以被设置为自然交配15。 </li><li>用细探针或小铲舀鸡蛋从下女性和/或她的肚子上。 </li><li>轻轻提起女性，回到她含鱼水系统适当标记的隔离箱。&lt;/ LI&gt; <li>加入50微升的紫外线失活精子溶液到蛋的离合器，并在室温下孵育30秒，在此期间，贴上标签对应的菜，以与分配给母本数量跟踪。 </li><li>加入1 ml的E3的蛋，并在室温下孵育1分钟。 </li><li>加入足够的E3解决方案，填补了中间的菜，然后将胚胎在28℃，随后孵化。注：除青霉素/链霉素（1％（V / V）含5000单位青霉素和每毫升5毫克链霉素青霉素-链霉素溶液）19至E3可以协助在单倍体胚的总体健康状况。 </li></ol> 5，观察和处理单倍体胚胎<ol><li>经过4-8小时，从盘取出未受精的胚胎，并用新鲜的E3解决方案取代了E3胚胎媒体洗剩余的胚胎。注：未受精的胚胎会显示一个白色的，不透明的外观或显示一个透明ppearance一个畸形的单细胞。 </li><li>直到孵化利益所需的时间点，然后利用在所需的屏幕化验或其他研究中的应用（次）。 </li></ol>

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The authors have nothing to disclose.

单倍体筛查是一项有用的技术来发现所需的早期发育阶段的必需基因。另外，从青鳉鱼单倍体细胞的培养已用于分离那些有许多研究应用和潜在的，这表明单倍体细胞可以用于其它类型的脊椎动物基因研究20,21提供了宝贵的未来地点ES样细胞系。该协议提供的涉及通过试管受精，用紫外灭活的精子进行生产单倍体斑马鱼胚胎的方法的示范。此方法可用于用诱变F1鱼，可以使用野生型，转基因或突变株为增强/抑制筛选进行执行前向单倍体屏幕。 虽然使用单倍体策略筛选时间被大大缩短相比，二倍体筛选，但是这也将需要几个月的时间才能完成。正如我们所记述先前的床，还有，通过使用一个单倍体模式，所有这些都可以在使左右任意数量的发育事件的当前知识新颖度有助于执行屏幕存在许多好处。单倍体屏幕的方法是更有效的隐性突变等位基因比二倍体的基于屏幕的因时间，空间，和有需要的鱼的数量的减少鉴定。然而，有几个陷阱，以单倍体屏幕。单倍体屏幕只能识别突变的基因是活跃在发展的最初几天，由于胚胎只能在有限的时间生存与单倍体基因组。那些在发展以后表达基因需要通过不同的方法，如一个二倍体屏幕来识别。此外，一些器官不正常的单倍体生物发展。有可能是解剖学异常，或发展的延迟时间，这可能导致该突变由单倍体筛选技术被错过。我Ñ ​​另外，鱼的某些菌株不以单倍体条件15发展为好。单倍体可以通过分级一系列的好，中，差的胚胎特征进行分类，其中A胚是最正常的，B到指定的胚胎与轴缺陷，但是一个明确的头部和尾部，和C / D指定胚胎无法辨认功能（群众细胞卵黄顶上球）15,17。在这些类别中的胚胎的比例变化由斑马鱼品系，以更优质的单倍体在特定遗传背景的15,17较为普遍增加的数字。由于这些因素，有必要找到斑马鱼的一个适当的菌株进行诱变和随后的杂交。在我们最近的经验，野生型蒂宾根株产生可行的单倍体筛选（ 如图3和图4），虽然历史上的斑马鱼研究人员已利用AB或AB *株单倍体experimentation 15。 除了上述这些挑战，不是所有涂底漆的成年雌性斑马鱼将应执行的挤压技术生产的离合器。例如，在用ENU诱变蒂宾应变女性工作时，大约有一半的女性产生经压缩的离合器，以及它们的一些部分（通常10-30％）是胚胎质量不好或不能受精。因此，要进行单倍体屏合一必须计划根据需要筛选基因组（每个女​​性代表一个基因组筛选）所需的数量，以提高至少两倍的鱼。无论这样的考虑，筛选以覆盖大量的基因组没有大规模动物设施的单倍体方法的好处是确实相当显著，如果分析方法是适合于单倍体状态。但是，也应指出的是，突变（S）在单倍体离合器确定的进一步研究是完全依赖于成功地养育Ñ​​异交的女创始人。正如任何屏幕上，&#39;打&#39;在筛查过程中初步确定必须重新确定的二倍体状态。 尽管使用了单倍体屏幕的缺陷，它已证明是在确定的突变体，已在深入分析了具有洞察力的涨幅，科学领域的15个非常成功的。单倍体屏幕可以被实现为调查脊椎动物发育的其他知之甚少流程。利用单倍体的增强和抑制的屏幕是获得更多的洞察和活动感兴趣的基因调控网络的方法之一。单倍体筛选技术也可以被用来识别增强剂和已分离的方法，如化学或插入突变或反向遗传学方法，如TILLING或TALENS突变体的抑制剂。总之，先前分离的突变体可以被诱变用ENU和筛选增强或s原突变体的表型uppressors。该突变体必须能够达到成年阶段，所以它需要有一个杂合的动物或弱纯合突变体鱼来执行此屏幕上，但在转基因的最新进展已经使研究人员能够绕开这个问题。学习如何操纵途径的发展可能会导致许多令人兴奋的可能性，包括扰动途径的前景来治疗的疾病状态。

编排脊椎动物发育的基本过程大致保守1。一个强大的方法，以确定基因与发展至关重要作用是隔离遗传突变导致的表型缺陷，在利益的过程。斑马鱼， 斑马鱼 ，是属于鲤科鱼的家庭，已经成为一种广泛使用的模式生物，脊椎动物的发育遗传学在过去的几十年中2小淡水硬骨鱼类的物种。斑马鱼的卵被外部受精，从而准许接近所述受精卵从受精3的发作。另外，在早期胚胎是光学透明的，从而使显影的可视化用一个简单的显微镜3。斑马鱼的个体发育迅速，与乳沟，原肠胚形成，许多结构，如心脏和肾脏，基本完成了开发3的第一天的器官的过程。 Ŧ他的时间从幼虫期到性成熟需要2-3个月，而成年人是小型脊椎动物长约3-4厘米，这么多的鱼可以保持在最小的空间2。此外，斑马鱼的成年人有较高的繁殖力，每个鱼能够生产每2星期几百个胚胎。这些属性都呈现斑马鱼的易处理性的正向和反向遗传筛选。斑马鱼具有保护高度在他们的解剖学，细胞生物学，生理学与更高级的脊椎动物物种，如哺乳动物2,4。事实上，最近的序列分析表明，斑马鱼基因组中含有同源近70％的人类基因5。这种保护使研究人员能够利用斑马鱼作为模式为许多人类疾病，包括胚胎和成年条件2,4。总而言之，这些因素使斑马鱼一个优秀的系统屏幕旨在确定基因的位置正常脊椎动物个体发育至关重要。 一些大型的屏幕已经进行了斑马鱼，并在发育基因6-8发现数千突变。斑马鱼成年男性是服从诱变和染色体改变可以与相对高的频率利用化学诱变剂乙基亚硝基脲（ENU）6,7或逆转录病毒插入诱变9产生。迄今为止，已有超过9,000遗传的突变已被创建，并且包括基因影响胚胎发生的几乎所有方面。斑马鱼的社区已经建立了这些突变体的银行集中在国际斑马鱼资源中心（或ZIRC）10，已经从世界各地收集约5,000突变和转基因的等位基因。许多这些突变进行了分析和克隆，让研究人员跨生物学科已被用来捉弄除了NUMERO有价值的信息通过发起与斑马鱼实验的见解，我们的细胞和生理途径。 尽管前进的屏幕已经确定的重要的基因数目可观，很多还没有意识到有关的基因调控网络，调解的过程万千。迄今开展的许多画面都没有达到饱和，从而推进以及反向遗传筛选仍然是一个基石，以识别和分析胚胎发育的机制。虽然有一些可以从任何单一的斑马鱼突变系中搜集到的巨大的见解，在该领域的主要限制因素，历史上曾参与了定位克隆的时候费时费力。出于这个原因，许多化学诱导突变的身份一直是个谜。然而，随着全基因组测序的方法，以促进快速克隆11-14近期问世，令人难以置信的高效识别基因突变是目前FEasible。 在斑马鱼中的典型正向屏幕是二倍体屏幕，可以三代6,7内识别隐性突变（ 图1中 ，左边的列）。这项技术涉及在父母（P）的男性的生殖细胞产生突变，然后交配这个男性与野生型女性。每个从该配合的第一代（F1）的后代将怀有因突变的父本基因组的染色体贡献的一个或多个独特的突变。 F1子代被提出，并远交与野生类型来创建F2家庭。在F2家庭，兄弟姐妹将实行野生型或杂合子携带者，并incrossed随机生成，这对于感兴趣的表型（S）分析，F3离合器。杂合子携带者的F3离合器将包含25％的野生型，50％杂合性，并且25％的纯合突变体鱼。这种多代筛选模式是有效的，但是一个主要的缺点是这种一pproach包括，用于在屏幕准备所需要的时间：至少1年独自鱼养殖的参与，因为每一代人需要3个月左右达到性成熟。这种类型的二倍体屏幕的其他限制的工作，空间和住房这些世代成本。 单倍体屏幕是一个替代方案，可用于识别，并随后分离出隐性突变使用类似的诱变策略15（ 图1，右列）。单倍体斑马鱼胚胎的生产被首次描述了30年前的16，和这段时间以来的正常二倍体斑马鱼的几天发展与单倍体染色体倍性的能力已经被用于众多的遗传研究。单倍体屏幕的主要好处是，这种方法不涉及募集F2家庭为了筛选隐性突变等位基因。相反地​​，F1代雌性为评估对象隐性突变的利益通过检查他们的F2后代在单倍体条件（ 图1，右列）的过程合度。整体，步骤，促使单倍体胚是相对简单的（ 图2）。准备精液处理所需的解决方案后，卵子从F1代雌性通过手工操作获得的，从而挤压（或“挤”）将鸡蛋从腹部。一旦卵获得，它们通过暴露于紫外（UV）灭活的精子受精体外 。紫外灭活精子是不能由于该短波长的UV交联的父系DNA的事实有助于可行的DNA的鸡蛋。然而，精子尚能触发蛋活动，并与合子发育相关的事件。经代谢活化，卵子将完成减数分裂II，并继续发展与各chromoso只有一个母体沉积副本我。正因为如此1N套数，胚胎是受隐性突变等位基因的发展后果，如果是存在于母体染色体。因此，每个F2单倍体离合器将包含50％的野生型和50％的突变的胚胎，如果母亲是一个完全渗透隐性等位基因的杂合载​​体。胚胎基因型的这种分布使得相对容易地在评价离合器为感兴趣的突变表型（S）的存在方面。如果这样的表型检测，F1代女创始人随后向远交野生型雄性斑马鱼来创建和筹集更多的杂合子携带者。因为这是没有必要提高多代履行屏幕，研究者可以节省大量的时间，人力和水族馆的空间，但仍然有调查基因组的基础上探讨女性F1的数目显著数量的权力。因此，单倍体屏幕是可行尤其在absenc发起的小型实验室或工程Ë投入大量资金。 但是，也有一些局限性和缺点，使用单倍体。首先，单倍体胚胎斑马鱼只能活数天，通常死于天受精后（DPF）15 3和5之间。单倍体斑马鱼的胚胎可以从基于几个特点二倍体加以区别，其中最重要的是一个短而粗壮的身体仍具有体节段15,17的正常数量。随着开发的进展，大脑表现出增加的细胞死亡和血液循环差，由2-3 DPF和水肿15,17与血池通常是相关联的。此外，单倍体不能夸大鳔15,17。不管这些形态差异，最主要的器官和组织形成，包括心脏，眼睛和脊索15,17。一个功能注意到对单倍体离合器就是它们含有各种表型的品种有缺陷的胚胎＆＃方面8212;整个斑马鱼品系的特征观察。因此，人们应该验证组织（S）和感兴趣的时间点显示出合理的正常发展，在野生型单倍体菌株，所以对在屏幕或其他研究项目的遗传缺陷进行评估的潜力。 尽管存在这些挑战，单倍体屏幕已成功实施，以确定必要在斑马鱼早期发育过程中的基因，和单倍体的协议已经确立社区资源，多年15-19。最近，产生单倍体鱼胚胎的过程已被用于研究人员创建单倍体胚胎干从青鳉鱼20,21（ES）细胞培养物。生产体外鳉单倍体胚后，胚被用来推导传代约15周，然后再5-8周，以产生单倍体细胞与纯克隆原代细胞培养胚胎干细胞特性15-19。鱼单倍体胚胎干细胞系的产生持有分析隐性表型在脊椎动物细胞谱系广阔的未来的承诺，并代表基因分析的新方法在未来几年。因此，单倍体鱼胚胎的一代生长在生物医学研究领域的应用。这个视频文章中提供的涉及生产体外单倍体斑马鱼胚胎使用基于既定协议15-19,22紫外线灭活精子的步骤的可视化演示。

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:364px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hank&#39;s Stock Solution #1</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">8.0 g NaCl, 0.4 g KCl in 100 ml ddH2O</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Hank&#39;s Stock Solution #2</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">0.358 g Na2HPO4 anhydrous; 0.60 g K2H2PO4 in 100 ml ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hank&#39;s Stock Solution #4</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">0.72 g CaCl2 in 50 ml ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hank&#39;s Stock Solution #5</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">1.23 g MgSO4-7H2O in 50 ml ddH2O</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;">Hank&#39;s Premix</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">Combine in the following order: (1) 10.0 ml HS#1, (2) 1.0 ml HS#2, (3) 1.0 ml HS#4, (4) 86 ml ddH2O, (5) 1.0 ml HS#5.&#160;&#160;&#160;&#160;&#160; Store all HS Solutions and Premix at 4 degrees Celcius.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Hank&#39;s Stock Solution #6</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">0.35 g NaHCO3 in 10.0 mls ddH2O; make fresh on day of use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hank&#39;s Final working solution</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">Combine 9.9 ml of Hank&#39;s Premix with 0.1 ml HS Stock #6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XX-Series UV Bench lamp</td>
			<td style="width:99px;">UVP</td>
			<td style="width:119px;">95-0042-05</td>
			<td style="width:364px;">Model XX-15S Bench Lamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">XX Bench Stand (Adjustable)</td>
			<td style="width:99px;">UVP</td>
			<td style="width:119px;">18-0062-01</td>
			<td style="width:364px;">Model XX-15 Stand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Embryo incubation dish</td>
			<td style="width:99px;">Falcon</td>
			<td style="width:119px;">35-1005</td>
			<td style="width:364px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">50 X E3</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">250 mM NaCl, 8.5 mM KCL, 16.5 mM CaCl2, 16.5 mM MgSO4 in distilled water. Supplement with methylene blue (1 x 10-5 M) (Sigma M9140) to inhibit contamination.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 X E3</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:364px;">Dilute 50 X E3 in distilled water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereomicroscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;">SMZ645; SMZ1000</td>
			<td style="width:364px;"></td>
		</tr>
	</tbody>
</table>

production of haploid zebrafish embryos by in vitro fertilization

斑马鱼已成为一种主流的脊椎动物模型是相关的科学研究的许多学科。斑马鱼特别适合于发育过程前进遗传分析，由于其外部受精，胚胎大小，迅速个体发育，和光学透明性 - 特性，使直接观察的活动范围从原肠胚形成到器官发生与基本的体视显微镜的一个星座。此外，斑马鱼的胚胎可以在单倍体状态下存活数天。 体外单倍体胚的生产是一种强大的工具，突变分析，因为它可以使隐性突变等位基因的鉴定存在于第一代（F1）雌性载体在亲本（P）生成下列诱变。这种方法消除了需要提高多个世代（F2，F3 等 ），涉及突变的家庭繁殖，从而节省了研究者的时间随着减少需要对斑马鱼克隆空间，劳动力和饲养成本。尽管斑马鱼已被用来进行着屏幕，在过去几十年里，出现了转基因和基因组编辑工具，稳步扩张。这些工具现在提供了大量的方法来创建细致入微的分析，为新一代的屏幕，可以用来进一步剖析了基因调控网络，推动脊椎动物个体发育。在这里，我们描述了如何准备单倍体斑马鱼胚胎。这个协议可以为未来的新颖单倍体的屏幕，如在增强子和抑制屏来实现，以解决发展的机制，在早期胚胎阶段形成过程和组织的广泛数量。

单倍体胚的生产可以实现识别隐性突变在F2时产生单倍体从成熟的雌性斑马鱼是诱变的父母的后代（ 图1）获得的。这样可以节省时间和空间相对于传统二倍体屏幕，其中研究人员需要提高F2家庭的二倍体后代，然后评估感兴趣的表型（ 图1）。 在该协议中的主要步骤在上述步骤1-5中所述，通过体外受精获得单倍体斑马鱼胚胎图式为流程图（ 图2）。这些步骤需要准备的解决方案和女性的暴露在男性荷尔蒙在交配坦克（第1天）吸，其次是精子和卵子的采购和体外受精 （2日），终于育成了单倍体（3-5天） （ 图2）。当COMbined与遗传筛选，在斑马鱼的共同诱变过程包括斑马鱼雄性野生型的成人以前暴露于诱变剂（ 例如 ，像ENU化学诱变剂，尽管其它程序，例如基于逆转录病毒的诱变可以实现这也便利的染色体缺陷克隆），其次为繁殖与雌性野生型创建诱变子代。这类制剂需要几个月的工作（约6个月）的基础上需要后野生型的世代时间，进行诱变，和后面的后续诱变F1 15,17,22。 考虑在采用单倍体的屏幕再大点是斑马鱼品系的选择。对AB *（AB星级）菌株已成为众所周知的生产单倍体胚胎比其他菌株15更好的整体性能和健康。然而，在图宾根派生应变孕育在我们的实验室保守党，我们最近观察到，已成功启用屏幕的肾脏系统（G.格拉赫和R.格特，未发表）的发育异常胚胎的功能。成年斑马鱼图宾根株男性由一系列的三个ENU诱变处理，然后和野生型杂交图宾根的女性产生的F1代进行筛选。 F1的女性被挤压，得到鸡蛋和这些F2卵子受精，用紫外灭活的精子。相比于野生型图宾根的父母（ 图3A）自然交配获得二倍体离合器，在这些单倍体图宾根株离合器胚胎显示的单倍体状态较短，树干粗壮特性（ 图3B，C）。进一步，单倍体离合器通常包括用于显示一系列缺陷，其中我们观察到，以及（ 图3B，C）中，这是已知的菌株内变化，如下面进一步讨论15,17胚胎兄弟姐妹。 </p&gt; 以前的研究已经建立了规范的分级系列，经过D对应等级A，进行分类的单倍体型缺陷15,17的范围内，另外单倍体也被提到的形容词好，中，差的质量的22方面。 A级单倍体，对应高品质的单倍体，是最正常的外观，用短而粗壮的身体，但总体表现出外观形态相似的二倍体15,17,22（以单倍体A级（标有比较二倍体的兄弟姐妹（四） A）兄弟姐妹， 图3B）。单倍体A级胚胎还制定适度的心包水肿，这是单倍体斑马鱼发育15,17,22（ 图3B）的典型特征。相较于A级单倍体，中间或所谓的B级单倍体胚（标记为B， 图3B，C）是通过进一步缩短或扭结/ TWIS的发展区别特德躯干，虽然头部和尾部的特征是清晰可辨15,17,22。最后，C级或质量差单倍体（也列在一些参考文献为C / D）15,17,22是非常有缺陷的，并在协会与卵黄球（标记为C， 图3C）显示细胞排列紊乱质量。甚至其中一脉相承，单倍体离合器在A，B和C表型的人会观察到的分布有所不同。例如，相对于从第二蒂宾根女，其离合器由众多C级单倍体后代，以及获得单倍体从一个女性的蒂宾根大学的单倍体离合器显示整体更好的发展，主要是A和B级单倍体后代（ 图3B）和B单倍体表型（ 图3C）。单倍体胚档次的同类菌株的变异已在AB和AB *应变鱼以前观察到的以及15。 尽管有这些铁道部phological差异，许多结构的器官进行比较正常的单倍体胚胎。尽管它们具有较短的身体躯干，发展像眼睛和心脏相对类似于野生型二倍体胚和细胞类型，如色素细胞（黑色素细胞和xanthophores）也可以评价15器官。此外，我们最近记录的鱼头，或肾胚胎，是1天受精后在野生型单倍体开发，类似于野生型二倍体。作为这方面的证据，鱼头肾细胞类型图案显示段的原型模式（ 图4）。另外，该改变鱼头发展，如解放突变，降低视黄酸产隐性突变的表型，显示了类似的模式在单倍体状态相比，二倍体状态（ 图4）23,24。这个观察是符合其他的衰退中它们以保存大量的突变，导致类似的表型在两个单倍体和二倍体状态，虽然这并不总是与所有突变的情况下，应牢记，如果评估这种类型的屏幕策略15。 许多组织和器官通常是不正常的单倍体。例如，单倍体显示在流通的缺陷，通常表现出的血池，这可能使他们的研究血管生成的某些方面能力的限制15。此外，已经注意到，单倍体可以有不规则耳发展，使得它们可以用于突变，防止耳泡形成完全被评估，但不适用于影响涉及这个结构15的形态发生微妙过程的突变。作为另一个例子，大脑形态异常的单倍体，从而单倍体筛选，以确定所需的大脑发育途径是有限的15。尽管有这些限制，我们一肾脏在发育单倍体状态（ 图4）nalysis加入这个机关的“遮”胚胎结构的列表。这一发现突出了一个单倍体屏幕的方法可以用来解剖肾祖图案的遗传成分，并增加了重点的情绪，巧妙屏幕的方法可以规避的单倍体胚胎个体发育的局限，发掘有价值的新突变模型来研究脊椎动物发育15。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51708/51708fig1highres.jpg" width="500" /> 图1原理图中的斑马鱼模型二倍体和单倍体屏幕策略。继亲代的诱变，F1代可提出并使用，也可以产生用于筛选的F3代的二倍体状态（左F2突变体的家庭）或直接屏幕编在一个单倍体的策略（右）。在一个单倍体的屏幕，性成熟的F1代雌性用于收集单倍体F2离合器进行基因分析。女性杂合子携带者被确定，如果大约一半的F2单倍体显示突变体的表型（红色胚胎）相比野生型单倍体的兄弟姐妹（黄色胚胎）。 <img alt="图2" fo:content-width="4in" src="/files/ftp_upload/51708/51708fig2highres.jpg" width="400" /> 图2。单倍体胚胎生产流程图。 体外受精产生单倍体胚胎的主要步骤是系统化的任务，第1天（粉红色框）进行准备精子的解决方案，并在第2天（橙盒进行总理成年雌性斑马鱼，任务）收集和紫外线灭活的精子，收集鸡蛋，受精蛋用紫外线灭活精子生成单倍体，然后重振女的母亲，和finallŸ任务在随后的日子里进行3-5（黄色框），当离合器培养至所需时间点（s）的观察和分析。 <img alt="图3" fo:content-width="3in" src="/files/ftp_upload/51708/51708fig3highres.jpg" width="300" /> 图3。二倍体和单倍体图宾根株斑马鱼胚胎的比较。 A）二倍体野生型胚胎是由自然产卵产生的，然后孵育，直到大约30 HPF，B，C）单倍体野生型胚胎体外受精从F1代雌性诱变用紫外线灭活的精子得到的离合器生产的，直到培养约30 HPF。单倍体展出了一系列的发育异常与二倍体野生型胚胎（黑色箭头，d来表示二倍体）进行比较。相对正常的单倍体有缩短，更多的股票的身体类似的评级制度一个A单倍体15（红色箭头，标记为A），而单 ​​倍体与严重畸形表现出严重截短体轴（紫色箭头，标记为B）对应于B级，最后C级（蓝色箭头，标记为C）的基础上出版说明15。所有的胚胎在2.5倍的放大倍率拍摄现场。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51708/51708fig4highres.jpg" width="500" /> 这包括在单倍体菌株蒂宾斑马鱼的鱼头胚胎肾器官的细胞类型的图4。发育构图。单倍体野生型显示细胞类型的正常模式中的胚胎肾结构（顶行）。胚胎肾由一对被称为肾分段功能单元组成。存在于肾单位的离散细胞类型的分割图案可以基于整个山被可视化<e米&gt;在基因转录所特有的分化肾细胞类型，其中包括标记的wt1b和肾病蛋白的足细胞（P）的原位杂交表达模式，该近曲小管（PCT）的标记slc20a1a，近端小管直（PST ）标记TRPM7，远侧早期（DE）标记SLC12A1和远端肾小管晚（DL）的标记SLC12A3。解放单倍体突变体胚胎（底行）显示减少足细胞，PCT和一个缺席PST，以及一个扩展DE和DL段，类似于二倍体胚胎库 18。胚胎被拍到在背视图，与前左侧，在10倍的放大倍率。

Watch this Scientific Journal Video about Production of Haploid Zebrafish Embryos by In Vitro Fertilization at JoVE.com

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 ...

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Biology

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

24.4K 次观看.  University of Notre Dame. 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.

视频: Production of Haploid Zebrafish Embryos by In Vitro Fertilization

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

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 ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...