简介小分子RNA的基因表达的重要调节，调节许多重要的生物过程活动都出现。超过700人的miRNA的发现至今，这并不奇怪，已与多种生理功能紊乱，如癌症和心脏疾病的 2,3的突变和不当的miRNA调控。最近的研究表明，大多数的miRNA结合位点的监管3&#39;UTRs 4,5，和一些研究人员估计，有数百个miRNA靶基因在细胞 6 。由于miRNA的生物的重要性，大量的存在，并为每个目标的大量的miRNA的，这是关键，研究人员能够可靠地识别内源性miRNA的目标，最好的平台上，适合高通量研究，。 miRBase，TargetScan，PicTar的1,6,7等，同时利用计算工具，是一个功能强大的方式来产生假定任何miRNA的表达目标的清单，预测尚未不够准确，8时单独依靠。因此，研究人员还利用实验方法来识别目标从头或验证的预测目标。许多球队都依赖稳态mRNA和蛋白水平的高通量的措施，以确定基因表达的变化时miRNA活性扰动 4,9,10 。这种变化无疑是生物学意义，是一个重要的第一步，但是衡量整体表达的变化之间的miRNA直接影响不区分成绩单和间接或下游信号效果。其他强大的工具，提供直接miRNA的基因相互作用的证据是染色质免疫沉淀（ChIP）战略，交叉链接argonaute蛋白（从而RISC复合物）的miRNA和mRNA的，虽然确定相互作用的伙伴不立即指示功能的影响这样一个具有约束力的事件5,11。因此，3&#39;UTR记者分析功能，提供一个miRNA在一个特定的非编码区的影响证据已经成为一个彻底的miRNA研究的重要组成部分。这种细胞为基础的实验数据计算目标预测或表达和蛋白质组学数据提供了有力的证据，特别是3&#39;UTR直接利益的miRNA调节。 在3&#39;UTR记者测定，从一个感兴趣的基因3&#39;UTR融合的荧光素酶报告基因的结束。如果3&#39;UTR是一个miRNA的目标，记者谈话内容的稳定性和/或它的翻译效率与功能的miRNA浓度的变化将改变。所以，不像成绩单只检测，记者分析，帮助找出两个稳定状态mRNA和蛋白水平的影响。记者系统也可以用来研究mutagenizing一个3&#39;UTR中的一个假定的miRNA结合位点，以确认它的确是必要的interaction8功能的影响。同时使用表达阵列，蛋白质组学技术，和argonaute芯片的大规模的研究提供了有价值的全基因组数据的实验条件下，基于单元的记者分析少数是最适合缩放研究miRNA的3&#39;小分子或其他库为基础的效应的高通量筛选，在数百或数千作为条件的非编码区相互作用可能成为必要。 许多科学家已申请报告基因检测技术的研究miRNA的3&#39;UTR相互作用。然而，与笔录各不相同的目标，多则上百的miRNA，它为研究人员很难利用一种高通量的规模强大的方法的想法，因为创造数千个不同的克隆，验证和优化的步骤3&#39;UTR记者载体往往成本过高。在努力使小型和大型3&#39;UTR记者访问的任何研究人员的研究，我们已经创建了一个高度优化的LightSwitch萤光素酶检测系统预先克隆人类3&#39;UTRs的全基因组的集合。记者系统还包括了有效的控制向量，合成miRNA靶700多记者构造，作为阳性对照使用，以及一系列标准化的实验协议。 使用下面的协议，你可以问一个特别的人类3&#39;UTR是否是您感兴趣的miRNA的目标。在这个工作流程中的关键步骤是实验设计，记者构造和miRNA模拟转染，荧光素酶报告分析，和数据分析。实验设计，重要的是若有所思地选择一个转染的细胞株，实验组和对照记者构造，和miRNA的模仿和控制非目标。常见的高转染ADHerent如HT1080，肝癌细胞株，HCT - 116，Hela细胞，293，和其他经常工作以及与本议定书​​。 实验设计的重要注意事项 LightSwitch萤光素酶检测系统是一个完全优化的记者系统，包括GoClone结构，利用荧光素酶基因的RenSP和LightSwitch萤光素酶检测试剂。使用的建议，以获得最佳的记者检测结果LightSwitch系统的所有组件。 开关GoClone记者构造与任何寡核苷酸/质粒共转染降低整体荧光素酶的信号，在一个非特定的方式。因此，重要的是，执行以下的转染组合:只有记者，记者+非目标控制的miRNA，和记者+特定miRNA。 我们empty_3UTR向量（强子，没有UTR）作为阳性对照转染效率，提供了一个荧光素酶的信号非常高的水平，并为阳性对照，模仿活动可以作为一种人工合成的miRNA靶记者载体。此外，我们建议使用GoClone 3&#39;UTR看家控制和随机控制，准确地分离序列特异性与非特异性的影响。 研究协议，并尽可能使主混音由于移液错误或蒸发，以减少你的数据的可变性。 该协议使用DharmaFect双核转染试剂（Dharmacon）。 第1天重要的提示:明智地选择你的细胞株。为了达到最佳效果，使用一个，被称为是转染的细胞株。我们还发现，一个miRNA的3&#39;UTR的记者可测量响应模仿是，在特定的miRNA高内源性水平的细胞株减毒。如果你不知道多少miRNA的细胞株表示，尝试SGG的合成小分子RNA的目标，间接措施的一个特定的miRNA在细胞以及通知您为您的检测的动态范围丰富。 1。种子细胞的转染时产生的≥80％汇合。 <ol><li>在96孔白色训练班板100μL总量的种子细胞。 </li><li>与此同时，种子细胞的适当数量的评估汇合在一个清晰的96孔板。 </li></ol>重要的秘诀1:汇流的关键是在模拟实验中实现了良好的效果。最佳击倒，细胞应为100％，转染时相连。 重要的秘诀2:使用低传代细胞，以取得最佳效果。已传过很多次的细胞往往产生嘈杂的转数据。 第2天制定GoClonereporter结构和miRNA，然后转染到的融合细胞。 2。准备构造<ol><li>在室温解冻GoClone构造（质粒DNA）。拌匀。 </li><li>离心机除去凝结从第。 </li></ol> 3。准备微RNA分子<ol><li>解冻后的小分子RNA（不针对特定的模仿和控制），在室温和离心机除去凝结上限。 </li><li>稀释到一个无RNase水2微米的工作浓度。 </li></ol>另外一个实验性的miRNA的模仿总是包括非靶向miRNA的控制。共同转染质粒和小RNA寡核苷酸低于转染的质粒单独的信号。 包括一个或多​​个阳性对照。考虑使用合成miRNA靶记者在SGG的目录向量数百。 包括一个或多​​个负面或非特异性控制。 SGG有包括3&#39;UTRs看家基因，随机片段和/或空（没有非编码区插入）控制的控制范围。一个或多个一起实验构造这些控件的使用将允许你为任何非特定的效果归属于您的特定miRNA的过度表达或控制非目标正常化。 4。准备转染<ol><li>对于每个转合并为一个单一的检测下列试剂: </li></ol> 混合1 <table border="1"><tbody><tr><td>个人GoClone记者: </td><td> 3.33微升</td></tr><tr><td>小分子RNA: </td><td>而有所不同* </td></tr><tr><td>无血清培养基: </td><td>至10μL </td></tr></tbody></table> * miRNA的有效浓度，可以从10纳米100纳米。为适当浓度的miRNA的制造商咨询。 重要提示 :不要添加太多的模仿。一个合理的范围内模仿浓度100uL反应在最后的10-50纳米。 执行至少有三个副本TE转染和每个治疗/ UTR的结合，包括额外的体积，让移液错误。 例如，乘以3.5单一的反应体积，以获得足够的转染3个复制的染井组合，包括额外的移液错误或蒸发。 对于每一个记者，设立以下复制:只有记者，记者+非定位控制miRNA和记者+特定miRNA。 设置针对非控制的同时，针对小分子RNA的转染。 <ol start="2"><li>化妆DharmaFECT DUO（Dharmacon）混合为每转如下: </li></ol> 混合2 <table border="1"><tbody><tr><td> DharmaFect铎: </td><td> 0.15微升</td></tr><tr><td>血清自由的媒体: </td><td> 9.85微升</td></tr></tbody></table> DharmaFECT铎要添加的量可能会取决于细胞系。 上述数额是适当HT1080细胞。 请咨询制造商的文档，以确定您的细胞系适量。 提示:记者人数乘以以上的卷，使一个大的转染混合物，复制和miRNA的数量进行测试。当计算转染组合准备，一定要包括少量的额外考虑到移液错误和蒸发。 <ol start="3"><li>允许DharmaFECT铎混合物在室温下孵育5分钟。 </li><li> 5分钟后，加入10μLDharmaFECT铎混合物（混合2）每个10uL质粒和/或miRNA的（1混合）准备管。每个管应包含每复制转20μL货量。 </li><li>每20分钟在室温下孵育混合物。 </li><li> 20分钟后，每管加80μL预热（37℃），不含抗生素，每复制完整的媒体共为100μL每复制转。一个深井块可用于许多复制和样品，同时准备。吹打向上和向下的混合解决方案。 </li><li>从孵化器中取出的种子板块。验证细胞至少有80％融合。 </li><li>小心吸管关闭从每口井的媒体。 </li><li> 100μL，每孔加入转染混合物（20uL DNA /试剂混合+ 80uL媒体）。 </li><li>广场板块的培养箱中培养过夜。 </li></ol>不要过分孵化。大多数模仿会产生真正的目标，在24小时内的显著成果。压制在48小时来衡量的水平可能会更大，但会以及良好的可变性。 模仿实验抑制剂的实验更容易。虽然大多数模仿将显著抑制真正的目标，在24小时内显著去镇压抑制剂，可以采取48小时内是可以衡量的，几乎总是不到的模仿效果明显。 第3天 注:荧光素酶检测，可进行即时或板块可能会被冻结在-80 ° C（冻结普遍增加细胞裂解和荧光素酶的信号）。冻结和解冻之前，你的细胞化验，以取得最佳效果。 记得让之前加入LightSwitch检测试剂室温下解冻冷冻细胞。 5。测量荧光素酶的活性与LightSwitch AssayReagents <ol><li>从孵化器中删除板，并带至室温。 </li><li>准备LightSwitch检测试剂（LS010套件，其他试剂盒大小的协议可能会在网上找到）: <ol><li>重新加入100μL底物溶剂冻干检测底物管100X基板。 避光，尽量减少在室温下的时间。 100X基板可以存储在- 20C 23周。为了达到最佳效果，使用新鲜的重组基板 。 </li><li> 10ML瓶检测缓冲​​液在室温水浴解冻，准备检测解决方案，并添加100μL重组100X基板之前使用。拌匀。 </li></ol></li><li>使用多通道移液器添加100μLLightSwitch分析解决方案（缓冲区+基板）直接向每个样品在一个白色的96孔板（含100uL细胞+媒体）。如果细胞生长在另一个板或瓶的格式 ，传输96孔白色板100μL总量（媒体或PBS）。 </li><li>盖板，保护光，孵育30分钟，在室温下，如果化验超过一个板块，错开除了检测解决方案，使每盘前30分钟阅读孵化。 </li><li>在每一个板LUMI以及读2秒nometer（SpectraMax L或同等）。 </li><li>计算计算每个特定miRNA在非目标控制（发光=特定miRNA /非定位控制）构建荧光素酶的信号比的击倒。看家，随机的，空的构造中使用的数据，以控制非- UTR，具体的治疗效果。 </li></ol>代表性的成果 3&#39;UTR目标靶向miRNA的存在表明荧光素酶击倒。 在HT1080细胞中let - 7A的3&#39;UTR的荧光素酶报告的活性测定击倒，每个记者的联合转染100ng 20NM - 7A让模仿或不针对控制建设，培育24小时，然后阅读发光记者信号使用LightSwitch萤光素酶检测试剂。 <img src="/files/ftp_upload/3343/3343fig1.jpg" alt="图1" /> 图1。 PUNC和ARID3B人类3&#39;UTRs - 7A让小分子RNA的目标 。PUNC和ARID3B基因从人类3&#39;UTR记者的信号让7A的microRNA存在显著撞倒在模仿，而家务和随机序列信号UTRs都没有。

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作者在这篇文章中出现的工具和试剂的开关柜的基因组学的员工。生产这篇文章是由开关基因组主办的。

我们创建了一个预克隆人类的3&#39;UTR记者构造的全基因组的集合，使活细胞中miRNA的3&#39;UTR相互作用的研究。在这里，我们已经表明了一个共同转染的miRNA与3&#39;UTR记者使用的荧光素酶报告系统的模拟重现性好，高通量的方法。 这种方法依赖于研究人员使用一个高度转染的细胞株和过表达技术的干扰miRNA的活性。研究人员使用不善转染的细胞株，可考虑使用我们的同伴慢病毒3&#39;UTR记者收集六西格玛（团3&#39;UTR Lenti GoClones）一起创建的。治疗与miRNA的细胞，模仿过度表达型实验的结果，并与任何此类研究中，研究人员必须考虑到，这可能会或可能并不代表实际的生理条件。为了解决过度表达的关注，研究人员可以使用除了以模仿的miRNA抑制剂的研究与两个不同类型扰动 12-14的互动。 一旦确定了可能的miRNA靶基因，研究人员可能希望mutagenize公认的miRNA结合位点，以确定是否网站功能互动的必要。他们可能还希望扩展到测试数百个不同的潜在目标UTRs（Boutz）。此外，如果有兴趣的miRNA是否影响成绩单稳定或翻译效率研究人员，荧光素酶报告数据可能会比较稳态转录水平的措施。在荧光素酶的信号的任何改变，不相关的，在转录水平的比例变化将指向对翻译效率的影响。最后，研究人员可以使用向量记者创建一个剂量反应曲线来比较不同UTRs的相对灵敏度在 14 miRNA的浓度的变化。

<table border="1"><tbody><tr><td></td><td> 供应商 </td><td> 产品编号 </td></tr><tr><td>白培养板（96孔坚实的底部） </td><td> VWR </td><td> 82050-736 </td></tr><tr><td>清除培养板（96孔）与盖</td><td> VWR </td><td> 353072 </td></tr><tr><td> LightSwitch萤光素酶检测试剂</td><td>开关</td><td> LS010 </td></tr><tr><td> DharmaFECT双核转染试剂（0.75mL） </td><td> Dharmacon </td><td> T - 2010 - 02 </td></tr><tr><td>铝箔板封箱胶带</td><td> E＆K科学</td><td> T592100 </td></tr><tr><td>透气板密封胶带</td><td> E＆K科学</td><td> T896100 - S </td></tr><tr><td>板光度计</td><td>分子器件</td><td> SpectraMax大号</td></tr></tbody></table>

identifying targets of human micrornas with the lightswitch luciferase assay system using 3'utr-reporter constructs and a microrna mimic in adherent cells

微RNA（miRNA）是基因表达的重要调节因子，在许多生物过程中发挥的作用。到目前为止，已经确定的700多名人类miRNA的每个有多达数百个独特的靶mRNA。识别是一个特定的miRNA的直接目标的mRNA的计算工具，表达和蛋白质组学分析和染色质免疫沉淀反应为基础的技术，提供了重要线索。此外，记者3&#39;UTR检测已成为一个彻底的miRNA靶标研究的重要组成部分，因为它们提供的功能的证据和定量特定miRNA 3&#39;UTR在基于单元的系统的相互作用的影响。为了使更多的研究人员利用3&#39;UTR记者分析和支持的规模化高通量水平等检测，我们已经创建了一个全基因组的集合人类3&#39;UTR的荧光素酶记者，在经过了高度优化LightSwitch萤光素酶检测系统。该系统还包括合成的miRNA靶记者结构，作为阳性对照，各种内源性的3&#39;UTR记者结构，和一系列标准化的实验协议使用。 在这里，我们描述个人3&#39;UTR记者构造共转染的方法与miRNA的模仿，效率高，重现性好，适合高通量分析。

Watch this Scientific Journal Video about Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells at 

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

微RNA（miRNA）是基因表达的重要调节因子，已被证明在许多生物过程中发挥的作用。为了更好地了解miRNA的UTR区的相互作用，我们已经创建了一个全基因组的3 UTR的荧光素酶记者的集合，搭配了一种新的荧光素酶基因的检测试剂，LightSwitch系统。

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 ...

identifying-targets-human-micrornas-with-lightswitch-luciferase-assay

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

36.1K 次观看.  SwitchGear Genomics. 微RNA（miRNA）是基因表达的重要调节因子，已被证明在许多生物过程中发挥的作用。为了更好地了解miRNA的UTR区的相互作用，我们已经创建了一个全基因组的3 UTR的荧光素酶记者的集合，搭配了一种新的荧光素酶基因的检测试剂，LightSwitch系统。

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

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

遗传学

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

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

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

生物学

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

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 ...

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 ...

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

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