This work was supported by NIH/NIEHS R03ES021581 (awarded to M.D.R.) and by the University of Rochester Environmental Health Center (NIH/NIEHS P30 ES001247).

1，飞文化，解决方案和胚胎处理装置的研制<ol><li>准备果蝇的网箱养殖。放置500 +配合所需的应变的苍蝇在装有10cm的葡萄琼脂平板和酵母膏的点群的笼子。在25°C湿度控制培养箱中保持文化。注意： &#160;凯奇文化需要一天的调理或两个获得一致的胚胎铺设图案。葡萄板的酵母膏是一次在早晨，一次在晚上调理过程中改变。 </li><li>准备的EPS。表面活性剂（椰油酰胺DEA和乙氧基化醇），在37℃暖解决方案移液器18毫升d-苎烯的一种玻璃闪烁瓶中装有一个小的搅拌棒。移液器各2表面活性剂（每5％的最终浓度）为1毫升的d-苎烯。调匀，取出搅拌棒。注：此股票的EPS解决方案是好的约2个月室温。该解决方案应在37℃下被加热并旋动至完全溶解，使用前的表面活性剂。 EPS和d-苎烯，应存储在一个玻璃容器，因为它们会溶解某些塑料随着时间的推移。 </li><li>准备胚胎dechorionation解决方案，孵化介质，染料和药物的解决方案。混合25毫升漂白剂用25毫升H 2 O和发生在浅盘。按照现有配方1,12制备改性的基本培养液（MBIM）和MBIM-T。制备盾并根据制造商的方案相M3细胞培养基和PBS（见材料清单）。准备通透性染料原液在10 mM的浓度在DMSO中。 </li><li>组装胚胎处理设备及用品： <ol><li>准备dechorionation和EPS治疗篮（ 见图1A）。切断一次性50毫升聚丙烯离心/文化管细齿锯的3厘米部分。使焊接表面齐平摩擦砂纸上的管段坚持到台式表面。焊接管部到Nitex尼龙筛通过熔化筒部的周缘在火焰和按压到网格上的玻璃板。晾凉剪掉多余的网格。注：纯粹的两侧和底部以便迅速而完全冲洗掉残余漂白剂和每股收益的各个步骤。 焊接步骤应在通风橱中进行。 </li><li>准备开发篮子。切断一个50ml的离心/培养管与盖的边缘齐平的顶部。除去并通过切出沿轮辋的中心孔和切口， 如图1B所示修改的上限。螺丝帽在网和在切断管段的线程和修剪多余的网格。注：上限包含在允许扩散到大型媒体定位在60毫米储菜（ 图1B&#39;）时，边缘的缺口。 </li><li>准备一个滑动室的部件。切DO膜的平方即大于滑动室开幕。适用于非常薄的层的真空润滑脂与开口的内唇缘。贴上DO膜进入封口它反对用挡圈油脂。通过削减其背部紧贴固定环修剪多余的DO膜。注：规格为滑动室可以在Kiehart 等[13]发现这个房间是不是市场上买到，由机加工车间需要定制加工。 </li></ol></li></ol> 2，分期，Dechorionation和胚胎的EPS治疗<ol><li>通过定时采集分期胚胎。设置一个新鲜葡萄/酵母板飞文化笼中的PM。允许在25℃下敷设1小时的胚胎放弃这个板块，并与新鲜的葡萄/酵母板在25°C取代2小时的后续发育铺设收集这些板块和地点在18℃培养箱中进一步发育分期。注：1小时发展在25°C等于2小时发展在18°C。在18℃下维持盈利通透晚期胚胎老化对25°C的效果可以看出，在图2。 </li><li> Dechorionation。轻轻冲洗胚胎关闭葡萄板到使用25°C的自来水和画笔网状筐。冲洗多余的酵母远离下自来水吹干篮下的胚胎。沉浸在篮下50％的漂白粉2分钟。彻底下的自来水流洗胚胎。注意：轻轻喷上间歇性使用塑料吸管的胚胎漂白剂溶液。而2分钟通常是足够的完整dechorionation，这个孵化时间应该由直接检查进行检查并进行相应调整。保持dechorionated胚胎浸泡在自来水中的篮子，并立即着手对EPS的一步。 </li><li>每股盈利治疗<ol><li>制备6 60mm的培养皿，在每个大约10毫升的PBS。在2.925毫升MBI溶解75微升准备的EPS摊薄每股收益M（1时40分）在与回旋50ml的玻璃烧杯中。注：从该混合物得到的白色乳液形式。 </li><li>从网格篮实验室擦拭底部吸干多余的水分。篮子沉浸在烧杯中稀释每股收益和漩涡立刻驱散胚胎在篮子底部的EPS解决方案。继续旋转运动，持续30秒。注：EPS稀释和曝光时间可以变化，以控制渗透性。建议最佳稀释每股收益和治疗时间凭经验苍蝇所使用的菌株成立。增加曝光时间60-90秒，有利于舞台12岁及以上的胚胎。 </li><li>删除篮子，吸干多余的走带EPS实验室擦拭。继续进行，在60mm的培养皿10mL的PBS 6的顺序洗涤。用塑料吸管轻轻喷胚胎用PBS各六个清洗的。继续染料和药物治疗的步骤。注意： 每股收益可出售倒入水槽。 </li></ol></li></ol>3，染料和药物透性胚胎的治疗<ol><li>染料处理<ol><li>加入5微升10毫CY5羧酸染料*的MBIM-T（50μM的最终浓度）至1ml在1.5ml微量离心管中，并涡旋混合。注：*染料的选择取决于下游分析。 CY5羧酸是有效利用固定和染色后续分析。罗丹明B是活胚胎的分析非常有用。罗丹明B的红色发射，其代谢产物1的绿色发光，可以提出一些并发症与使用荧光下游应用。药物或毒素可以加入到染料溶液中开始治疗在此阶段，或以限制药物/毒素治疗的脉冲在这个阶段。应小心处理和处置的药物和毒素根据MSDS和环境安全标准。 </li><li>从网篮转移胚胎到使用画笔的染料溶液。盖上管并倒转重复，以确保胚胎自由浮动在悬浮液中的染料溶液。放置管上的章动摇杆15分钟，在室温下进行。 </li><li>从章动器取出试管，让胚胎定居。除去染料溶液，用细吸管和替换用1ml MBIM-T洗。反转管完全重新挂起胚胎。让胚胎定居并重复三个MBIM-T洗。从最后冲洗除去所有MBIM-T和继续孵育步骤。 </li></ol></li><li>培养胚胎发育<ol><li>移植胚胎的两院之一：开发篮子或滑动室。注：开发篮优选更长的发育阶段，并且是必需的，如果后续的固定和染色步骤将被执行。滑动室是最佳的分辨率更高的时间推移成像，并且是最有效的短发育阶段（ 例如 ，早期胚胎的事件）。药物或毒素，可向培养基中加入各种浓度和胚胎ð才有发展可以实时用活的胚胎或端点处使用标准的固定和免疫染色的协议来监测。 </li><li>发展篮<ol><li>用70％乙醇喷出清洁发展篮子，用去离子水彻底冲洗并抹干与实验室擦拭。准备6毫升孵化介质与药物或毒素的所需的浓度。将发展篮子中型60毫米盘回吐照顾下的网格基础不是陷阱气泡。注意：两个介质常用的有：MBIM单独或MBIM/M3在50:50混合物，后者是更有效的较长的开发周期。 </li><li>透转移胚胎到网面筐的底座采用了画笔。轻轻地从周围的水库唧与媒体的胚胎。分散的画笔将胚以使它们在网格上的一个单层。注意： 继续显微成像和评估公关的通透性和生存能力特点eparation（见第4步）。 </li></ol></li><li>发展中的滑动室<ol><li>逆转滑动室和施加一个小珠的真空润滑脂在开口的周边。把150微升培养基中的药物或毒素在开口内溶解氧膜的表面的期望的量。注意：两个介质常用的有：MBIM单独或MBIM/M3在50:50混合物，后者是更有效的较长的开发周期。 </li><li>透转移胚胎到媒体下降的画笔。分散使他们定居在下降中膜的胚胎。 </li><li>小心运用25毫米的圆形盖玻片了开幕从而扁平化的媒介。轻轻按下沿盖玻片的周边，形成与油脂密封。注意： 继续显微成像来评价制剂的透化和活力的特性（见步骤4）。 </li></ol></li></ol></li></ol> 4，鉴定出的透性活胚胎fication <ol><li>确定透胚胎。根据落射荧光观察胚胎以配备有数码相机的显微镜。采集图像（在蓝色波长来确定配置文​​件蛋黄自发荧光）的使用固定显微镜和相机设置多个领域。 </li><li>确定基于相对荧光强度胚胎通透。使用立体显微镜具有大的工作距离，以容纳篮并允许胚胎的操作。显微镜应该装有一个可编程XYZ载物台，落射荧光照明和数码相机。图像照顾到记录曝光和每个胚图像的阶段位置参数，以使相同的胚胎的重新评价在稍后的时间点的胚（见代表性的结果在图3中）。注意： 染料吸收的模式将取决于所用的染料，曝光和胚龄的长度变化。广泛跨越单一制剂染料吸收是典型的，并反映在透化的程度的变化。 </li><li>确定可行的胚胎。标志着透胚胎的位置后，继续与胚胎发育在室温或25°C。返回篮子或滑动室的显微镜。观察下蓝色通道的荧光，并获得在先前确定的透胚胎蛋黄自发荧光的形象。根据卵黄分布的正常进展评估活力（见兰特等人 1和具有代表性的结果在图3中）。注意： 用明视野显微术观察到的胚胎的其他形态的特征可以被用于评估生存能力。建议建立渗透性，这与生存能力兼容的电平根据经验与每个染料和用于油炸食品的应变确定。 </li><li>评估透活胚胎药物或毒素的效果。 </li><li>胚胎多项式ssed与上述协议都准备了多种常规分析，药物或毒素暴露以后。几种不同类型的分析被认为是在下面的讨论，其中包括在活胚胎直接观察形态，以及后固定染色分析。这两种方法都是通过使用活体染料（ 例如 ，GFP），揭示基因表达模式和细胞谱系和形态学访问增强。 </li></ol>

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The authors have nothing to disclose.

上述方法概括的手段来获得可行的果蝇胚胎都可以访问的跨越广阔的发展范围小分子治疗。这个方法引入了新颖而简单的结论，即在18℃下老化使胚胎后期胚胎的通透性具有相同功效的以前只在早期胚胎中看到。此外，用远红染料CY5羧酸作为渗透性指标的已被证明有效的定位后的应用程序，并且不具有可用于揭示发育表型的常规的红色和绿色的荧光标记物产生干扰。这些发现显著推进的EPS?...

果蝇胚胎仍然是一个首要的模型为开发2基本机制的调查。是通过允许基本上任何基因操作在任何时间点，并在所有发展中的器官的分子遗传工具的广泛支持这一强大的模型。小尺寸的果蝇胚胎的形态发生，发展迅速，广泛的特性使其成为首选的遗传筛选，其中许多已经发现根本发展途径3,4模型。在果蝇胚胎大量的表型进行了表征，并容易解释，往往提供确定负责异常性状潜在的分子遗传机制的一种手段。 从历史上看，在果蝇胚胎模型的一个缺点一直是引入小分子对胚胎组织的难度。这一障碍已对限制：1）我们ING已知的生物活性小分子作为探针询问发育机制以及2）使用这个既定的模型来评估未知的小分子致畸或药理活性。作为结果，在果蝇胚胎的筛选潜力已经得到充分利用的小分子活性的表征。 1）透蛋壳和2）显微注射：输送小分子的果蝇胚胎可以用两种方法来实现。本文介绍了，很容易在传统的果蝇实验室的设置来执行垫款通透的方法。但应注意的是，在微注射方法，与微流体技术的最新进展也有助于引入化合物对胚胎5,6的方法。将分子引入到胚胎是防止由蛋壳7的蜡质层。 果蝇蛋壳由五层组成。从内向外它们是：卵黄膜，蜡质层，内层绒毛膜层，endochorion和exochorion 8。三个外绒毛膜层可以通过在稀释的漂白胚胎的简要再现被去除，步骤简称为dechorionation。暴露的蜡质层然后可以通过暴露于有机溶剂，如庚烷和辛烷7,9，渲染dechorionated胚胎可渗透受到损害，而它仍然装在底层卵黄膜。然而，使用这些溶剂中的介绍，由于其毒性和对调节其强透化作用，这两者都在胚胎存活率9,10鲜明的负面影响的难度复杂化。 通透的使用成分称为胚胎透溶剂（EPS）的一种方法，以前已经描述1。这种溶剂包括d-柠檬烯和植物衍生的表面活性剂，使溶剂是misciblE使用水性缓冲液。 d-柠檬烯和溶剂稀释至所需浓度的能力的低毒屈服于产生渗透胚胎具有高生存能力1的有效方法。然而，有两个内源性因素继续带来限制的应用程序。首先，胚胎表现出异质性，透气性后每股收益处理，即使小心地保持着密切的发育分期。其次，胚胎早于约8小时已被证明难以通透，与后产蛋11时发生的蛋壳硬化一致。 这里描述的是在EPS方法的进展：1）协助查明和分析近透完全相同的胚胎，即使在固定和染色步骤已经执行，2）使胚胎的通透性在后期发育时间点（&gt; 8小时，阶段12岁及以上）。具体地，应用一个远红染料，CY5羧酸，记载了作为透气性指标，它在开发过程中和甲醛固定后仍然存在于胚胎。此外，它表明，在18℃下培育的胚胎保持在蛋壳的EPS敏感的状态，使后期的胚胎（12-16级）的通透性。 这些进步克服了前面提到的限制，对EPS的方法。因此，该应用程序将提供与调查，介绍景点小分子胚胎在不同的发育时间点，同时保持活力的一种手段。

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a method of permeabilization of drosophila embryos for assays of small molecule activity

果蝇胚胎一直是强大的实验室模型，以求澄清该控制发展的分子和遗传机制。遗传操作这一模式的易用性已经取代药物的方法是司空见惯的其他动物模型和基于细胞的检测。在这里，我们描述了一个协议，它使应用小分子显影果蝇胚胎的最新进展。该方法详细步骤，克服了蛋壳的抗渗性，同时保持胚胎的生存能力。在广泛发育阶段的蛋壳通透性是由应用程序先前描述的d-柠檬烯胚胎透溶剂（EPS 1），并通过老化胚胎在降低的温度（18°C）前处理实现的。此外，利用一个远红染料（CY5）作为透化指示剂进行了说明，这是与涉及标准的红，绿流感下游应用兼容orescent染料在现场和固定的准备。该协议适用于使用生物活性化合物来探测发育机制以及为研究旨在评估未知的小分子致畸或药理活性的研究。

胚胎处理装置， 如图1所示 ，以协助可视化在上述议定书“自制”的操作装置。结果如图2所示说明了他们的能力是由EPS为发展后期阶段的饲养透胚胎在18℃下的强大作用。此条件是在协议步骤2.1应用。该CY5羧酸染料以显示不同层次的渗透性通常出现在EPS处理的胚胎的功效被认为是在图3中 。在蛋黄的色素分布的发展的动力学也可见于图3中 ，揭示?...

Watch this Scientific Journal Video about A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity at JoVE.com

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

引入小分子到显影果蝇胚胎提供了用于表征的新化合物，药物和毒素的生物活性，以及用于探测基本发育途径的巨大潜力。本文所述的方法概括了战胜自然障碍，这种做法，扩大了果蝇胚胎模型的实用步骤。

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

a-method-permeabilization-drosophila-embryos-for-assays-small

Research

JoVE Journal

Biology

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

12.3K 次观看.  University of Rochester School of Dentistry and Medicine. 引入小分子到显影果蝇胚胎提供了用于表征的新化合物，药物和毒素的生物活性，以及用于探测基本发育途径的巨大潜力。本文所述的方法概括了战胜自然障碍，这种做法，扩大了果蝇胚胎模型的实用步骤。 

视频: A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...