Name: a method of permeabilization of drosophila embryos for assays of small molecule activity
Uploaded: 2014-07-13T14:00:00
Description: Watch this Scientific Journal Video about A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity at JoVE.com

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）...

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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

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

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

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

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