We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

注:吞吐量这种检测系统取决于可用的HTS实验室设备。该协议是由在Tecan自由EVO 200机器人，它在96孔格式进行液体处理容易。小型化到384孔格式也是可能的。机器人液体处理系统是为了在所有的实验步骤保持在无菌条件下放置在层流罩。如果没有液体处理自动化是可用的，该协议可以很容易地适合于低吞吐量的格式。 初筛 1.第一天:准备和种子细胞注:种子细胞，得到80％汇合在测定荧光素酶的活性，在接种后，即 ，48小时的时间。 <ol><li>重悬胰蛋白酶CHP134-mycn3UTR神经母细胞瘤细 ​​胞的2×10 5个细胞/ ml在RPMI含有10％FBS和1％L-谷氨酰胺的浓度。为27片制备至少250毫升<html>细胞悬液考虑到液体处理，波谷死体积和反复移液步骤。倒入细胞悬浮到无菌的贮存器并将其放置在所述机器人处。 </li><li>放置9条形码白色平底96孔板和一个盒上的机器人台200微升无菌吸管提示。各分注步骤之前混合细胞悬浮液通过移液以避免细胞在重力下沉降。分配75微升细胞成9板各孔，以得到每1.5×10 4个细胞的最终密度良好。 </li><li>盖上盖子的板和离开他们的保育箱的外面30分钟以使细胞沉降到井的底部。这将最大限度地减少边缘效应。 </li><li>重复，以制备27片的总数量的步骤1.2和1.3的两倍。 </li><li>将板在37℃和5％CO 2孵育24小时。 </li></ol> 2.第2天:治疗与库化合物的细胞ntent"&gt;注意:频谱收集小分子文库包含2000化合物在10毫在DMSO中的浓度设置在8行和10列的25个96孔板上的第一个和最后列中的孔是空的控件。 。化合物筛选试验一般进行的1-10μM化合物浓度8。这里所描述的筛查方案修复2μM的工作浓度和24小时的试验结束点。 <ol><li>解冻9化合物文库平板在室温下。 </li><li>准备两槽用无菌PBS及PBS-0.5％DMSO的解决方案，并将其放置在机器人的办公桌。每个库板，准备和标记相应一个稀释盘 - 聚丙烯U型底96孔板具有至少400微升一个工作容积。填写列2的每孔稀释板的398微升无菌PBS的使用液体处理器的固定秘诀11。填列1和12用50μl无菌PBS在为了再生在对照孔中的车辆的浓度（0.02％）0.5％的DMSO。 </li><li>将两个库板的相应标记稀释板，两箱50微升无菌枪头和机器人桌子六个单元板。揭开细胞板。 </li><li>吸取2微升相应的稀释板10毫米原液从两个库板块之一拌匀。这将导致在50微米的中间化合物的浓度。使用同一套技巧，取3微升50μM稀释3条形码白96孔板的细胞。这种稀释方案导致2μM工作的每个浓度测试化合物。 注:为避免不必要的操作与技巧，使用全套96提示从一开始，尽管库板仅包含80化合物。这是可能的，因为在第一和库板的最后一栏是空的。 </li><li>更换枪头，重复圣EP 2.4的第二个库板。 </li><li>覆盖细胞板，并将它们返回到培养箱24小时。 </li><li>对于其余的库板重复步骤2.3-2.6两次以上。 </li></ol> 3.第3天:执行萤光素酶检测注:在进行萤光素酶测定法中，也能够与基于刃天青还原，以便从每个孔9得到细胞存活率索引的细胞生存力测定法复它。复用萤光素酶和活力测定测定结果减少荧光素酶信号的，但是，可以忽略，如果细胞提供足够高的水平的萤光素酶的活性。荧光素酶活性可以通过各种商业和自制10,11-萤光素酶测定试剂稳定发光信号来检测。 <ol><li>平衡所选择的重组荧光素酶试剂至室温。确保音量足以让所有的检测板。 27板s170毫升萤光素酶试剂将需要考虑到液体处理，波谷死体积和重复的移液步骤。倾萤光素酶试剂成贮存，并将其放置在机器人处。 </li><li>从孵化器中取出9板的细胞，将它们放在机器人的办公桌，发现，等到平衡至室温。添加50微升每孔平板的荧光素酶试剂的由板。在板之间，引入一个延迟等于一个板的发光读数时间。这将确保所有的板被从试剂加成相等的时间间隔内进行测量。 </li><li> 30分钟温育后，将第一板中的光度计，测量短暂摇动步骤之后发光。重复所有板块。记录每盘的条形码，并将其关联到相应的数据文件，以避免来自大量处理板所产生的错误。 </li><li>重复两次，其余18板与细胞步骤3.2-3.3。 </LI&gt; <li>收集剩余的荧光素酶试剂，并将其储存在-20℃。 </li></ol> 4.第4天:数据分析<ol><li>过程实验数据使用所有样品的归一化，以对板载体处理对照的平均值。对于每个库化合物计算平均值X和SD超过一式三份。 </li><li>选择上调和下调通过设定X的阈值±K＆times SD，其中，平均值X和SD被计算在所有测定处理后的值，而k的命中是一个定义的常量。 </li><li>选择任意的阈值切断&lt;时减少荧光素酶信号的最有可能与化合物毒性相关媒介物处理的对照的20％。丢弃低于该阈值的命中将显著降低了一些在反筛选假阳性命中。 注:代替控制为基础的正常化，实验数据可被处理施加Z分数或其健壮类似物B·S芯12。此外，可用12命中选拔替代的统计方法。 </li></ol> 5.反筛查注意:被确认和评价的特异性通过反筛选在初级筛选中鉴定的化合物（标记为"命中"）。 <ol><li>樱桃采摘从存储在单个2D条码管的等分所选命中，创造新的"打板"，节省了相应的布局。不要忘记留下自由位置的控制。 注:在没有一个有效的樱桃采摘系统，一种可能测试对照和3&#39;非编码区承载细胞并联在初级筛选。在这种情况下，特定​​的选择与只依赖于引入的3&#39;非编码区作用的化合物将初筛消除反筛选的需要之后是可能的。然而，在两种细胞系的平行筛选将增加一倍的检测成本。 </li><li>继协议初筛（第"1天:准备和种子细胞"），为每一个"打板"准备三个96孔板每个CHP134-mycn3UTR和CHP134-CTRL稳定的细胞。 </li><li>继协议初筛（第"2日:治疗用化合物库的细胞"），使用每个打板治疗3 CHP134-mycn3UTR三CHP134-CTRL板在2μM工作浓度。 注意:尽管此处所描述的反画面中一式三份以2微米的单次剂量进行的，这可能是有益的替代标准重复和在3种浓度测试命中10倍稀释液从2微米至20纳米。应用这种方法，不仅允许以确认主画面的数据，而且还以获得在主命中初步剂量​​ - 反应数据，最小化假阳性率。在这种情况下，不同的分析管道应该是APPLI编处理实验数据和确认命中。 </li><li>继协议初筛（第"3日:执行萤光素酶检测"），测量所有CHP134-mycn3UTR和CHP134-CTRL板荧光素酶的活性。 </li><li>下列的协议初筛（参见"第4天:数据分析"），从规格化处理的孔的实验数据，以对板媒介物处理的对照的平均，并计算平均值和SD上重复。对于所测试的各化合物，计算荧光素酶活性在CHP134-mycn3UTR的倍数变化与CHP134-CTRL细胞。选择任意倍数变化下调取舍的&gt; &lt;0.01 1.5和意义p值。 注:在所描述的测定法设置命中特异性的决定因素参数是萤光素酶活性的一个显著倍数变化在CHP134-mycn3UTR与CHP134-CTRL细胞。如果需要的话，我们也可以通过在本身同时进行细胞活力测定评估化合物的毒性lected化合物。这可能是特别有益的，如果初级命中计数器筛选以剂量响应分析。对于单剂量，我们的经验表明减少荧光素酶活性很强的相关性与细胞活力下降6。因此，在靶细胞以及对照细胞减少荧光素酶活性可以被认为是化合物的毒性，在所测试的浓度的指标。 </li></ol>

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The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

对于基因表达的很长一段转录调控被认为发挥了重大的，如果在控制蛋白质的生产不是唯一的作用。越来越多的证据，但是，表明转录后调控贡献之多，如果不超过，转录调控，以确定细胞蛋白质丰度1,2。基因表达的转录后调控更加复杂和精细的比起初的想法。事实上，转录后调控的所有各个阶段都出现了调控，包括mRNA加工，本地化，营业额，翻译3以及新描述的可逆RNA甲基化4。从一些转录后调控过程可能受到影响，测定下面描述着重于那些涉及mRNA的3&#39;非翻译区（3&#39;非编码区）。 3&#39;UTR广泛影响mRNA的命运通过特定的RNA结合公关互动为主oteins和非编码RNA，以它的调节序列和/或二级结构5。因此，小分子改变这些相互作​​用或干扰的上游信号转导途径将转向，调节蛋白质丰度的平衡。这种治疗方法是用于人类疾病的量存在证据表明疾病相关基因进行转录后调节，从而代表了药物干预的潜在目标特别重要的。因此，系统通过用在高通量形式的转录后控制机制的干扰，允许mRNA水平"调节剂的筛选可成为潜在的新的治疗方法的鉴定有价值的工具。 所描述的基于细胞的报道基因测定允许对能够经由依赖于它的3&#39;端非编码区的机制来调节的mRNA命运的化合物的鉴定。它由几个主要的Components（ 图1），并且需要几个预备步骤来验证该测定的可行性。首先，所述报道构建体被设计为包括3&#39;从感​​兴趣的基因的UTR。的3&#39;端非编码区融合到报道基因的末端，如萤火虫荧光素酶（ 图1）。通过插入的3&#39;端非编码区施加的任何变化在转录后控制机制将改变该嵌合转录物的稳定性和/或翻译效率，从而导致不​​同的荧光素酶的水平。因此，改变荧光素酶活性的服务感兴趣的基因的受影响的转录后调控的间接测量。该系统的第二组分是控制报道构建，表达相同的报道基因，但不包含任何3&#39;非编码区相同的表达质粒。两个报告质粒可以设计在实验室使用常规的分子生物学的协议或从商业来源购买。合适的细胞系的选择是用于测定施工的关键。其中细胞系是最佳的模式取决于很多因素，包括从像最大的相似之处病理像可用性，transfectability和生长特性只是实际问题的临床需求。重要的是，前，继续进行1应该确保融合的3&#39;端非编码区的报道基因对嵌合转录物的水平的预期效果。没有来自3中的荧光素酶信号显著差异的&#39;UTR轴承和对照报道构建体瞬时转染在所选择的细胞将指示该3&#39;端非编码区是未官能此细胞模型中，提示输入搜索替代的。对于高通量格式中，3&#39;端非编码区，轴承和控制荧光素酶报道构建体应当稳定地整合在所选择的细胞系。使用稳定整合在瞬时转染细胞系是优选的有几个原因。这将扩大的蜂窝模型，包括难以转染的细胞系的选择，降低在从转染效率的波动所产生的数据的可变性，消退的费用。此外，当瞬时转染的细胞经常超负荷的DNA质粒导致系统的饱和度。在这些设置在记者表达的进一步增加可能是具有挑战性的，导致对潜在的上调化合物的灵敏度下降。 最后，当所有试验组分均成立，它是移动到高通量形式，以确定该测定， 即可行性前关键的，如果荧光素酶活性可以确实上调和下调经由插入3&#39;特异性UTR。最好控制将是报道通过感兴趣的3&#39;非编码区来调节mRNA的稳定性或译小分子。如果有这些是不可能的，替代的控制模仿所希望的效果被接受。这些可以是miRNA的或已知通过结合感兴趣的3&#39;端非编码区，以发挥它们的作用的RNA结合蛋白。评价这种控制前的初级筛选不仅表示显影测定法的功能，而且还允许估计在高通量形式的动态范围，灵敏度和性能。 使用所描述的协议，我们筛选了2000化合物库6。神经母细胞瘤，婴儿期最常见的颅外实体肿瘤，被用作生物模型和MYCN癌基因，其扩增的3&#39;端非编码区强烈预示神经母细胞瘤的不良结果，作为目标基因7。 图2列出了实验布局。筛选被分为三个运行与27板筛选一次运行该批处理大小。本批27片包括光谱采集库板N.1-N.8 + n.25（第一轮），N.9，n.16 + n.25（第二轮），并n.16，n.24 + n.25（第三轮），每个板筛选一式三份。因此，一个库板（n.25）所有三种运行使得可能相互比较的运行范围内测定。以下协议描述了一个单，一式三份测试9库板运行。

<table><tbody><tr><td>Culture plate 96-well White</td><td>PerkinElmer</td><td>6005688</td><td></td></tr><tr><td>StorPlate 96-well U bottom (dilution plate)</td><td>PerkinElmer</td><td>6008190</td><td></td></tr><tr><td>100 ml disposable trough for reagents</td><td>Tecan</td><td>10 613 048</td><td></td></tr><tr><td>300 ml disposable robotic reservoir</td><td>VWR</td><td>PB12001301</td><td></td></tr><tr><td>Robot tips DITI 50&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038607</td><td></td></tr><tr><td>Robot tips DITI 200&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038617</td><td></td></tr><tr><td>Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td><td>Thermo Scientific</td><td>3711</td><td></td></tr><tr><td>ONE-Glo Luciferase Assay System&amp;nbsp;</td><td>Promega</td><td>E6120</td><td></td></tr><tr><td>RPMI</td><td>Lonza</td><td>BE12-918F</td><td></td></tr><tr><td>PBS-1x, w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Lonza</td><td>BE17-516F</td><td></td></tr><tr><td>L-Glutamine 200&amp;nbsp;mM</td><td>Lonza</td><td>BE17-605E</td><td></td></tr><tr><td>FBS</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>The Spectrum collection (compound library)</td><td>MicroSource Discovery Systems</td><td>N/A</td><td></td></tr><tr><td>Tecan Freedom EVO200 robot&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr><tr><td>Tecan F200 multiplate reader&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr></tbody></table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

转录和转录后调控对基因表达产生深远的影响。然而，普遍采用的基于细胞的筛选试验集中于转录调控，而实质上旨在启动子靶向分子的鉴定。其结果是，转录后机制在很大程度上未覆盖由基因表达的靶向药物的开发。在这里，我们描述了，并在确定能够修改它的化合物旨在调查的3&#39;非翻译区（3&#39;非编码区），在其mRNA的命运的调制的作用的基于细胞的测定。该测定法是基于使用含有感兴趣的基因稳定地整合入一个疾病相关的细胞系的3&#39;非编码区一个萤光素酶报道构建体。该协议被分成两部分，与最初的焦点上的初级筛选旨在治疗24小时后影响萤光素酶活性的分子的鉴定。该协议的第二部分介绍了反筛选必要区分化合物特别是通过3&#39;UTR调控荧光素酶的活性。除了详细协议和有代表性的结果，我们提供关于该测定的开发和上内源靶的命中（多个）的验证重要的考虑因素。所描述的基于细胞的报告基因分析将帮助科学家识别分子通过依赖于3&#39;UTR转录后调节机制蛋白质水平。

使用所描述的方法，我们筛选了2000-化合物库的转录后控制机制的潜在调节剂通过MYCN基因的3&#39;非编码区施加。 图3示出了初级筛选由单个库板例举的结果。显示为媒介物处理的对照的百分比荧光素酶信号，通过测量荧光素酶活性与单个库板的化合物处理CHP134-mycn3UTR细胞的一式三份板获得。正如预期的那样，多数化合物对荧光素酶活性没有影响由接近100％基线值所示。命中（明...

Watch this Scientific Journal Video about High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes at JoVE.com

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

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 ...

high-throughput-screening-for-chemical-modulators-post

Research

JoVE Journal

Biology

9.4K 次观看.  University of Trento. 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’ UTR.

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

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

科研

遗传学

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

发育生物学

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

生物工程

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

癌症研究

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

免疫与感染

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

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, ...

生物学

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...