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&#039;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

Research

JoVE Journal

Biology

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

36.0K 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

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

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 ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

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.