我们感谢S.伯克哈特，C.霍夫曼和西蒙娜Gräßle优秀的技术援助，并感谢M. FERG帮助数据分析。我们也感谢S. Rastegar对稿件批评。我们承认由Studienstiftung DES deutschen Volkes（以兆瓦），东风集团（DI913/4-1）和亥姆霍兹计划BioInterfaces在KIT资金。

1。准备化学图书馆的工作稀释预稀释药物库进行测试的化合物（ 例如，FDA批准的药物，恩佐生命科学）为E3（5 mM氯化钠，0.17毫米氯化钾，0.33毫米氯化钙2）用机器人液体处理工作站（ 如多探头二，8针，处置末端适配器，珀金埃尔默）。合适的浓度在3％DMSO中，例如 40微克/毫升，但是这将取决于所用的库的类型。对应于下文中所述的步骤的多探头II的设置示于表1中 。在表中描述的协议允许四等分板平行的准备。 <ol><li>股票库（2毫克/毫升的DMSO）为深孔板（ 如 96孔存储板，圆形井，0.8毫升，ABgene）的移取10微升等分。列1和12应当留空 - 这些将接收的正和负板内对照。 </li><l<html> I&gt;分注490微升的E3中，用1％DMSO与多探头II的库的制备等分试样。稀释与固定针不进行处理的技巧。 </li><li>加10微升的DMSO进入塔1和10μl的5mM的地塞米松溶液（在DMSO中）到第12列作为板内对照。 DMSO在所有孔中的浓度为现在的3％，在阳性对照孔中的地塞米松浓度为E3 / 3％DMSO的100μM的。 </li><li>用DMSO性粘接密封片密封板。存储板在-80℃直到使用。 </li></ol> 2。记者鱼养殖收集GRE组之间的交配自然产卵胚胎：吕克·转基因鱼。提高胚胎（不超过60）在含有补充了杀真菌剂亚甲蓝1毫克/毫升到4天后受精（DPF）在孵化器在28°C的E3媒体9毫米培养皿定期更换的E3媒体。 标题“&gt; 3。制备荧光素酶培养基（E3L） 通过加入DH 2 O含有荧光素粉末的小瓶中制备50mM的水溶液荧光素储备溶液。此储备溶液可以贮存于-80℃下进行几个月。稀释的荧光素库存成E3中至0.5mM的最终浓度，以获得E3L介质。 4。幼虫分配到96孔板<ol><li>通过切断大约5毫米针尖，并简要燃烧的尖锐边缘，制造1毫升吸管尖端与大口径。 </li><li>从几个十字架从培养皿小心把它们倒在烧杯池幼虫。采收约50幼虫在同一时间轻轻浇他们筛（0.25毫米直径的孔径）上，并立即筛放入​​一个小培养皿（直径5.5厘米）充满E3L媒体。 </li><li>用大口径的枪头，转移225微升介质中含有每一个幼虫的白井96孔培养板（OptiPlate，珀金埃尔默）。 </li><li>密封板的粘接密封板（ 例如 TopSeal-A，珀金埃尔默），并培育它们在28℃过夜。这可以防止预孵育记录的生物发光的瞬时变化除了荧光素，其也发生在培养的细胞19的现象后，立即。 </li></ol> 5。药物治疗<ol><li>删除包含该库从-80°C冰箱约4小时使用前的工作稀释板。轻轻涡旋板并简要地旋转它在离心机中在板的底部收集的液体中。 </li><li>取下幼虫板粘接密封片。 </li><li>用手工操作的96通道移液装置（ 例如清盘，施泰因布伦纳）吸取药液的上下的3倍。然后分装25μL的化学库的工作稀释成含以上幼虫（与示例中的井，这结果在4微克/毫升在E3用0.3％DMSO）的终浓度。准备10（最低：5）复制板中，每库板的幼虫。 </li><li>密封板的粘接密封片及带条形码标签标记每个板。 </li></ol> 6。发光录音<ol><li>把含有幼虫板成的EnVision读取器的生物发光增强的发光灵敏度（PerkinElmer）上的堆叠单元。或者，使用机器人培养箱系统，如在与读取器组合使用的用于哺乳动物细胞培养物的筛选系统。 </li><li>记录的生物发光，使用读卡器设置类似于表2中的Envision读数器描述两天。适应测定的重复数，以板中的运行次数，以匹配所需要的运行时间。 </li><li>在运行结束后，检查板的死幼虫的存在，以评估样稿一般毒性ounds。 </li></ol> 7。数据分析在使用自定义脚本的统计编程环境R被执行的所有数据分析。 <ol><li> AUC计算 为了确定化合物的活化GC信令不论其动力学性质的，确定所记录的发光痕迹（生物发光的原始计数与时间的关系）作为筛选参数的曲线（AUC）下的面积。 AUC值是近似梯形规则（参见图2a和a&#39;）。 </li><li>正常化 记录，以确保较高的正态的数据（ 图2b和b&#39;）的变换的AUC值。然后正常化数转换AUC值对每个库板与强大的Z评分法（见20）。这个归一化结果在较所有数据点的中值的标准偏差的数值。以这种方式，系统误差，如跨运行变化，从数据中删除。该数据可以通过绘制平均值鲁棒Z值的副本的每个孔（ 图2c和c&#39;）被可视化。 </li><li>质量指标<ol><li>热图 为了形象化板相关的影响，绘制为每块板的数据作为使用如 GPLOT包21的heatmap.2功能的热图。以这种方式，边缘效应或化合物结转可以容易地检测（ 例如， 图3a与图3b相比）。从进一步分析中排除次优的平板上。 </li><li> ROC曲线 要确定灵敏度的检测和特异性，计算受试者工作特征（ROC）曲线，例如使用在7.2（ 图3c）确定的Z评分值中华民国Bioconductor的包22的帮助。随后，确定此立方米的AUCRVE。的AUC值接近1表示高灵敏度的测定的和特异性。 </li></ol></li><li>命中鉴定 为了优化截断值对活性化合物的鉴定，确定尤登指数。为了计算指数，从假阳性率的ROC曲线增加强大的Z值的临界值的（FPR）减去估计的真阳性率（TPR）。采取稳健的Z评分最高约登指数（TPR减FPR）为分界点。该TPR / FPR可以通过检测库中已知活性化合物的阳性对照孔或进行估计。当使用阳性对照孔，确保它们符合预期的打击力量。 </li></ol> 8。复检<ol><li>通过处理幼虫与从不同的供应商得到的化合物的系列稀释液，使用相同的记录建立如上所述的重新评价阳性的命中化合物。真实命中应该引起一个发光LSO在该测定中，理想的剂量依赖性。 </li></ol>

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我们什么都没有透露。

我们在这里的工作流程和数据分析，化工生产屏幕使用GRIZLY检测18测量GC活动在体内 。该法具有质量控制的特点和措施，性能可与传统的基于细胞的屏幕相媲美，其在高通量的设置使用的一个重要优势。此外，然而， 在体内测定法也检测到不能访问在体外筛选化合物。这是通过点击其中的激素原孕烯醇酮的存在下举例说明。因此，GRIZLY测定延伸屏幕旨在GC信号传导活性的范围。 与我们的测定法体内作用的检测的重要性也由我们用有机锡污染物的DBT和TBT 18得到的结果示出。 DBT，但不TBT，已被证明能抑制GC信令在哺乳动物细胞培养物，并且我们观察到的相同的斑马鱼细胞培养物中表达ing的GRE：吕克记者。然而重要的是，在GRIZLY测定中，三丁基锡化合物表现出抑制活性，在GC途径，因为它可以由幼虫代谢被转化为DBT。这种抑制作用观察到已经在贸易技术壁垒的环境有关​​的浓度。这些结果进一步说明在检测的基础上脏器间串扰和代谢中的活的动物的化合物的修饰化合物的效果的GRIZLY测定的电位。他们还强调对环境监测的GRIZLY检测的应用潜力。因此，内分泌干扰效应可以在受体信号，其中所述干扰物干扰引起的由GR激动剂如地塞米松GC信令活性水平进行研究。另一种可能性是，研究孕烯醇酮刺激GC合成化合物的效果。检测的高通量质量应该允许环境样品库的快速筛选。 利用荧光素酶作为AReporter基因可以检测信号的活性23的高动态范围。事实上，测定的灵敏度，能够检测渗透胁迫诱导的GC生产从单一幼虫18。实现了与萤光素酶报道的高时间分辨率可以让我们跟随信令活动一段时间，这使得能够同时分析数据的动力学。 对于某些应用程序的一个潜在缺点是有限的适宜空间监视记者的系统。荧光报告系统可能更适合用于需要监测的幼虫的某些区域的检测。然而，这样的测定法可以从较低的敏感性遭受由于受激发光，这也带来了对荧光化合物的检测限的背景效果。此外，他们可能会提供，因为荧光蛋白23的一般稳定性更高少动力学拆分。此外，他们目前在影像设备，数据分析和数据存储，可能限制其使用更小的研究实验室方面的挑战。 在设置了GRIZLY分析作为基础的微孔板检测可以很容易地集成到典型的筛查工作流程。该测定是很容易适用于也由于其简单的操作和数据分析更小的研究实验室。在同一时间，它允许自动化的显着程度， 例如通过移液机器人或胚胎的胚胎通过分拣装置24,25自动分配自动药物的应用。简单的读出不需要自动筛选显微镜或复杂的图像分析软件，但提供了一组丰富的研究信号通路的时间和数量方面的数据。 综上所述，我们提出了一个一步一步的协议，用于GC相对便宜，功能强大和易于处理药剂筛选试验信令活性在体内和实时性。该法允许根据检测的化合物的GC 在体内作用的决心信号没有检测到细胞培养。在众多应用中的试验都是如对糖皮质激素的信号，对糖皮质激素的合成和信令活性的内分泌干扰物影响环境监测，以及化合物的筛选在GC上的信令或小说中的这种调节体内不必要的影响遗传效应的测定重要的信号转导通路。

糖皮质激素（GCS）是由肾上腺这期间的应激反应和代谢1的调节起着重要的作用产生的类固醇激素。 GC的结合细胞质糖皮质激素受体 ​​，核受体超家族的，结合后，转运到细胞核2（GRS）。在这里，它们可以例如抑制转录通过与其他转录因子（阻抑）干扰或通过糖皮质激素应答元件（GRE的，反式激活）激活基因转录。由于它们的抗炎特性，天然的和人造的GC被广泛地在许多疾病，比如哮喘或关节炎3，用于治疗的药物。然而，特别是长期使用的GC可能导致严重的副作用，包括糖尿病和青光眼4。因此，针对GC上可能更有利的治疗效率和耐受性信号的新化合物十分抢手船尾呃。重要的是，在培养的细胞中观察到配体效应可以不同于见于体内 5。这种影响可能会与传统的细胞培养获得的偏差结果为基础的制药筛选试验。化工体内筛选的斑马鱼模型启用最近成为焦点，因为它允许的效应在细胞培养6,7无法检测的决心。 激素信号通路也受到环境污染物。所谓内分泌干扰物（EDCs）影响各种激素的调节过程8。因此，繁殖和水生生物的性分化可以通过与雌激素样活性物质进行调制。近日，有关人士提出，代谢紊乱可能在环境中的9挂内分泌干扰物。一个通道由这样的“代谢受阻”靶向的是糖皮质激素的通路，这也被牵连在异种对发育和免疫功能的10,11抗生素的效果。然而，随着大量的可用信息化合物与性类固醇激素作用的干扰相比，相对鲜为人知的是，通过GR介导的内分泌干扰作用。因此，需要工具，使体内监测GC的信号污染物的影响。 斑马鱼一直是一种流行的模式在发育生物学和较近期也引起了研究人员从其他领域，包括内分泌学12。与其他硬骨鱼类相比，斑马鱼的GC信令系统是更类似于哺乳动物的，因为斑马鱼的基因组中仅包含1 GR基因，而不是重复的受体在许多其他鱼类13-15。此外，下丘脑-垂体-肾上腺轴功能已经5天的斑马鱼幼虫，从而增加应对压力的内源性GC生产14,16-18。 我们最近所产生的转基因斑马鱼线，GRE：吕克，它允许进行GC信号活性的体内和实时18的监控。该生产线进行下一个最小的TATA盒启动子和四个concatemerized GRE的（ 图1a）的控制的荧光素酶报告基因构建。 GC诱导生物发光可以从单一的GRE来衡量：吕克幼虫在96孔板体内过长时间存放。这GRIZLY测定（为“糖皮质激素反应性的体内斑马鱼的萤光素酶活性”），可以以许多不同的研究领域，如应力研究，环境监测，和药理学的屏幕18的使用。我们能够探测到的内生增长皮质醇从单一的幼虫渗透胁迫后发育过程中可以遵循的响应的成熟。此外，我们可以监测有机锡的气相色谱信号的影响s表示需要代谢的幼虫。重要的是，该行能够检测在环境相关浓度的这些效果。最后，在一个导频屏幕测定灵敏和特异的检测用气相色谱活性的化合物由化学文库，包括刺激内源性皮质醇的生产中的幼虫一种化合物。在这里，我们描述了一个详细的协议使用GRIZLY分析化学的屏幕。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Buffer composition + reagents</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>Carl Roth GmbH &#38; Co KG</td>
			<td>A994.2</td>
			<td></td>
		</tr>
		<tr>
			<td>FDA approved drug library</td>
			<td>Enzo Life Sciences</td>
			<td>BML-2841-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Biosynth</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>E3</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Instruments</td>
		</tr>
		<tr>
			<td>Multiprobe II</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>8 channel, equipped with disposable tip adaptor</td>
		</tr>
		<tr>
			<td>Liquidator 96</td>
			<td>Steinbrenner Laborsysteme</td>
 <td></td>
			<td>hand-operated 96-channel pipette</td>
		</tr>
		<tr>
			<td>EnVision XCite Multilabel Plate Reader</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Equipped with stacker automation, temperature control, barcode reader and enhanced luminescence detector</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Plasticware + consumables</td>
		</tr>
		<tr>
			<td>96-Well Storage Plate</td>
			<td>ABgene</td>
			<td>AB-0765</td>
			<td>round well, 0.8 ml</td>
		</tr>
		<tr>
			<td>Cover films,</td>
			<td>ratiolab</td>
			<td>6018412</td>
			<td>self adhesive, DMSO resistent</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Steinbrenner Laborsysteme</td>
			<td>LRF-200L</td>
			<td>for liquidator 96, 200 &#956;l, low retention</td>
		</tr>
		<tr>
			<td>TopSeal-A</td>
			<td>PerkinElmer</td>
			<td>6005185</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005299</td>
			<td>white opaque 96-well microplate</td>
		</tr>
		<tr>
			<td>Barcode Labels</td>
			<td>PerkinElmer</td>
			<td>1608182</td>
			<td></td>
		</tr>
		<tr>
			<td>filtered polypropylene IsoTip pipette tips</td>
			<td>Corning</td>
			<td>S058.4809</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Animals</td>
		</tr>
		<tr>
			<td>GRE:Luc fish</td>
			<td>N/A</td>
			<td>ZDB-TGCONSTRCT-120920-1</td>
			<td>available at the European Zebrafish Resource Centre EZRC, Karlsruhe</td>
		</tr>
	</tbody>
</table>

a chemical screening procedure for glucocorticoid signaling with a zebrafish larva luciferase reporter system

糖皮质激素应激激素及其人工衍生物被广泛用于药物来治疗炎症，但与糖皮质激素长期治疗可导致严重的副作用。所需的测试系统以搜索影响糖皮质激素信令体内的新化合物，或确定化合物对糖皮质激素信号传导途径不想要的效果。我们已建立了转基因斑马鱼测定法，其允许在体内和实时糖皮质激素信号传导活性的测量中，GRIZLY测定（糖皮质激素反应性的体内斑马鱼的荧光素酶活性）。荧光素酶为基础的检测检测具有高灵敏度和特异性，包括那些需要代谢或影响内源性糖皮质激素的生产效果的化合物对糖皮质激素信号传导的影响。我们在这里进行化学屏用这个方法一个详细的协议。我们描述了数据采集，标准化和分析，把重点放在质量控制和数据可视化。该测定提供了一种简单，时间分辨，和定量的读数。它可以操作作为一个独立的平台上，但也很容易集成到高通量筛选的工作流程。另外，它允许超越化学筛选许多应用，如内分泌干扰或压力的研究环境监测。

在以前的出版物18，我们筛选的640 FDA批准的药物库中筛试点评估GRIZLY检测的性能。这个库包含12 真正的GC，从而使我们能够确定的敏感性测定的特异性和质量的措施。 图2示出的原始数据的分析和归一化从该屏幕截取的典型例子。从塞米松控制的典型结果以及示于图2a中 ，示出了由数据提供的时间信息。这发生在治疗后约12小时的生物发光峰值缓慢下降超过使用梯形法的AUC值以下36小时测量。 图2a&#39;突出了近似。这种计算是进行了全井和各技术重复。对数转换的结果示于图2b &lt;/ STRONG&gt; 和b&#39;。在这里，阳性对照的平均值绘制在一个正常的QQ图。登录变换导致的数据点由红色对角线表示的理论正态分布值的较大近似。的变换，实现了数据的正常性增加了后续分析的统计力量。最后， 图2c和2c&#39;显示的健壮Z分数归一化上的板到板变异性的影响：用不同的板（ 图2c）所得到的不同的基准值被归一化的鲁棒Z分数积在图2c&#39;。 鲁棒Z分数值可用于通过可视化横跨板的撞击的分布，以确定在数据的系统误差， 图3a示出一个热图的库板，其中一个例子，其中强大的Z分数缬氨酸UE被标绘彩色编码到井的位置。一个容易识别柱12与中板阳性对照。阳性得分图书馆化合物被认为在井F8和，尽管弱，E2。 图3b示出的板，其中一个系统误差的存在。一个人观察了一系列化合物，有超过数列递减行A和E 的Z-score值。无化合物在这些井是在重新测试呈阳性反应。由于井用这种下降GRIZLY测试活动放在沿等分库板时，移液机器人经过的路径，这种模式是指示在自动移液过程中积极的化合物的结转。 的高强度Z-score规范化连同已知的选区在图书馆的存在也使我们能够计算受试者工作特征（ROC）曲线屏幕18。 图3c显示了估计值的积交配真阳性率对估计假阳性率不同的高强度Z分数的临界值。估计真阳性率被定义为真阳性的命中被发现在一给定的鲁棒Z分数的截止值，该值的百分比（在此，存在于库中的已知糖皮质激素）。相应的估计的假阳性率表示非正化合物的百分比击中在此截止值。这条曲线的AUC是0.95，这表明具有高灵敏度的屏幕和优良性能测定和特异性。的AUC曲线也被用来通过计算约登指数为不同的强大的Z值来估计命中识别的最佳临界值。我们确定了稳健的Z值的1.49为具有最高约登指数。此临界值是表示在图2c&#39;作为黑线从大的化合物的分离命中。 在我们的屏幕18，我们能够确定存在于库中几乎所有已知的选区。只有未检出三12 善意的GC。中的醋酸美仑孕酮的情况下，这是一个假阴性结果，由于这种化合物测试在重试阳性在更高的浓度比在库中使用的1。另两个GC为阴性更有重新测试。皮质甾酮是在啮齿类动物的主要GC，但不是在鱼或人1，而该前药强的松可能不会通过幼虫系统代谢良好。有趣的是，还两种药物没有注释为选区分别在屏幕标识，其中一个不能在重试（安体舒通，盐皮质激素受体拮抗剂）证实。其它化合物，孕烯醇酮，是在类固醇的生物合成，这是由肾上腺转化为皮质醇前体。事实上，随着孕烯醇酮治疗刺激皮质醇生产幼虫18。所有这些结果证实了高表现ormance测定的：只有一个假阴性和一个假阳性的化合物是主要的屏幕结果之间，和10的GC信号刺激活性的11证实化合物已经确定在主屏幕上。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50439/50439fig1.jpg" /> 图1的分析和筛选程序的工作流程的一般原则。一）GRIZLY分析报道构建计划。荧光素（黄色）的表达是由一个最小启动子（P 分 ，橙）和4 concatemerized GRE元件（（GRE）4，白色），这是由配体激活的GR结合的同二聚体（GR，蓝色）进行控制。红色框表示TOL2转座的网站，便于整合构建成基因组中。粉红色的方块代表一个聚腺苷酸化位点（PA）。转基因幼虫携带这con结构体被放置在不透明的96孔微量滴定板中进行生物发光测量。 二）萤光素酶反应的方案。萤光素酶催化荧光素向氧化萤光素，从而导致发光（hν）的氧化。反应需要也ATP和Mg 2 +的，这是由幼虫提供。 我的PP，焦磷酸。 三）工作流程的画面。工作原液稀释从制备的FDA批准的药物库在96孔板中，一列载板内的阴性对照（仅DMSO，绿色）和含有阳性对照组（地塞米松，红色）（库设计）一列。 GRE：吕克幼虫分布在96孔板（5-11复制），并与库化合物（新药申请）处理。生物发光的痕迹都记录了生物发光读者两天（数据收集）。生物发光的痕迹都集成（AUC计算），对数转换和强大的Z-得分归一化（的归限）。归一化的数据被用于确定质量度量和对于命中识别（数据分析）。选择命中都在试验（复试）重新测试剂量依赖性活性。 <a href="https://www.jove.com/files/ftp_upload/50439/50439fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50439/50439fig2.jpg" /> 图2。从一个FDA批准的药物库中的屏幕数据的数据分析说明。 a）由阳性对照孔。 一&#39;）代表跟踪曲线（AUC）下的面积是近似梯形法则。用于计算梯形显示在不同深浅的红色二）正态QQ图的阳性对照孔（y轴）的日志转换前的原始AUC值与标准正常人群（x轴）的。B&#39;。 ）<html>正态QQ图对数变换后的AUC值的。一个正常的分布是通过对数转换数据点的线性表示。日志三）情节转换原始数据在屏幕上进行测试的所有化合物。蓝，糖皮质激素善意 ;强大的Z-score规范化后的画面数据的灰色，所有其他化合物C&#39;）剧情。系统误差，例如板间的变化已经从数据中删除。红色线表示的正极板内对照的平均值±SEM。黑线：命中识别临界值由约登指数优化（ 图3）来确定。转载自14日批准的ACS。 <a href="https://www.jove.com/files/ftp_upload/50439/50439fig2large.jpg" target="_blank">点击这里查看大图</a> 。 0439fig3.jpg“/&gt; 图3。屏幕效果。 A）热图画面效果。化合物库的强大的Z评分值绘制成相应的孔位置。在塔12中的阳性对照，以及在井F8和E2 2阳性的命中化合物是可见的。一个板显示文库制备用移液机器人在正化合物的结转二）热图。活动的渐变是沿可见采取的机器人移液路径。 三）受试者工作特征（ROC）曲线屏幕。估计真阳性率（左手y-轴）和假阳性率（x轴）作图增加强大的Z值的临界值（颜色编码，右侧y轴）。所得曲线的AUC值接近1，表明良好的检测性能。转载许可14，2012 ACS。 四）表的示化合物活性（YELLOW）或无效（蓝色）在主屏幕（prim.）或复试。 善意糖皮质激素并不总是复检（白色）。重绘从14日批准的ACS。 <a href="https://www.jove.com/files/ftp_upload/50439/50439fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td colspan="4"> 常规设置： </td></tr><tr><td colspan="4"> 步骤1.1 </td></tr><tr><td> 程序 </td><td>类型： </td><td colspan="2">单液</td></tr><tr><td></td><td>模式： </td><td colspan="2">井喷</td></tr><tr><td></td><td>省却每次抽吸： </td><td colspan="2"> 1 </td></tr><tr><td></td><td>优化速度： </td><td colspan="2">是的</td></tr><tr><td></td><td>气隙系统： </td><td colspan="2"> 10微升</td></tr><tr><td></td><td>交通： </td><td colspan="2"> 0 </td></tr><tr><td> 冲洗/清洗 </td><td colspan="3" width="426"> 无 </td></tr><tr><td> 吸气 </td><td> 设置类型 </td><td> 值 </td><td> 备注 </td></tr><tr><td></td><td>位置</td><td> B7，B10，B13，B16 </td><td></td></tr><tr><td></td><td>吸卷。 </td><td> 10微升</td><td></td></tr><tr><td></td><td>速度</td><td> 200微升/秒</td><td></td></tr><tr><td></td><td>液体追踪</td><td> 60％ </td><td></td></tr><tr><td></td><td>吸液高度</td><td>实验设备默认</td><td> 22.28 </td></tr><tr><td> 豁免 </td><td> 设置类型 </td><td> 值 </td><td> 备注 </td></tr><tr><td></td><td>职位</td><td> D7，D10，D13，D16 </td><td></td></tr><tr><td></td><td>免除卷。 </td><td> 10微升</td><td></td></tr><tr><td></td><td>速度</td><td> 200微升/秒</td><td></td></tr><tr><td></td><td>液体追踪</td><td> 100微升</td><td></td></tr><tr><td></td><td>点胶高度</td><td>实验设备默认</td><td> 17.01 </td></tr><tr><td colspan="4"> 步骤1.2 </td></tr><tr><td> 程序 </td><td> 类型： </td><td colspan="2"> 试剂 </td></tr><tr><td></td><td>模式： </td><td colspan="2">浪费</td></tr><tr><td></td><td>省却每次抽吸： </td><td colspan="2"> 3 </td></tr><tr><td></td><td>优化速度： </td><td colspan="2">是的</td></tr><tr><td></td><td>气隙系统： </td><td colspan=“2”&gt; 15微升</td></tr><tr><td></td><td>交通： </td><td colspan="2"> 0 </td></tr><tr><td colspan="2"> 冲洗/清洗 </td><td colspan="2"> 无 </td></tr><tr><td> 吸气 </td><td> 设置类型 </td><td> 值 </td><td> 备注 </td></tr><tr><td></td><td>位置</td><td> A4 </td><td></td></tr><tr><td></td><td>吸卷。 </td><td> 3×490微升</td><td></td></tr><tr><td></td><td>气隙</td><td> 15和0 </td><td></td></tr><tr><td></td><td>速度</td><td> 200微升/秒</td><td></td></tr><tr><td></td><td>液体追踪</td><td> 400％ </td><td></td></tr><tr><td></td><td>吸液高度</td><td>液体表面</td><td></td></tr><tr><td> 豁免 </td><td> 的S型<html>廷</td><td> 值 </td><td> 备注 </td></tr><tr><td></td><td>位置</td><td> D7，D10，D13，D16 </td><td></td></tr><tr><td></td><td>免除卷。 </td><td> 490微升</td><td></td></tr><tr><td></td><td>气隙</td><td> 15和0 </td><td></td></tr><tr><td></td><td>速度</td><td> 200微升/秒</td><td></td></tr><tr><td></td><td>液体追踪</td><td> 100％ </td><td></td></tr><tr><td></td><td>点胶高度</td><td>实验设备默认</td><td> 17.01 </td></tr></tbody></table> 表1中。设置多探头二。表1中的协议描述了4片（每96孔）的制备方法。该板位于上板的适配器中的位置处由机器人（ 例如，B7，B10，B13，B16）中指定的工作区域。 <html> 1“FO：保持together.within页=”总是“&gt; <tbody><tr><td colspan="4"> 常规设置： </td></tr><tr><td colspan="2"> 实验重复次数 </td><td colspan="2"> 依赖于运行长度 </td></tr><tr><td colspan="2">板数</td><td colspan="2">无限</td></tr><tr><td colspan="2">温度控制</td><td colspan="2"> 28°C </td></tr><tr><td colspan="2"> 协议 </td><td colspan="2"> 计算 </td></tr><tr><td></td><td colspan="3"> 美国绥96（CPS）读 </td></tr><tr><td></td><td>测量时间</td><td> 2.5秒</td><td rowspan="3">串扰校正</td></tr><tr><td></td><td>板和检测器之间的距离</td><td> 0毫米</td></tr><tr><td></td><td>辉光（CT2）修正系数</td><td> 0％ </td></tr></tbody></table>内容“&gt; 用于在屏幕表2中。的EnVision设置。

Watch this Scientific Journal Video about A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System at JoVE.com

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System | Biology  | Video

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

我们描述了利用斑马鱼幼虫糖皮质激素的应激激素信号的化学筛选系统的程序和数据分析：糖皮质激素响应在斑马鱼体内的荧光素酶活性（GRIZLY）检测。该法灵敏，特别是通过需要代谢或影响内源性糖皮质激素的生产检测化合物对糖皮质激素信号传导的影响。

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

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11.8K 次观看.  Karlsruhe Institute of Technology - Campus North. 我们描述了利用斑马鱼幼虫糖皮质激素的应激激素信号的化学筛选系统的程序和数据分析：糖皮质激素响应在斑马鱼体内的荧光素酶活性（GRIZLY）检测。该法灵敏，特别是通过需要代谢或影响内源性糖皮质激素的生产检测化合物对糖皮质激素信号传导的影响。 

视频: A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

转染RAW264.7细胞用荧光素酶报告基因

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...