我们要感谢加里·诺兰博士从斯坦福大学提供的凤凰GP包装细胞系用于生产逆转录病毒颗粒。我们感谢罗杰钱永健博士在加利福尼亚大学圣地亚哥分校提供TD番茄。我们还要感谢圣迭戈州立大学流式细胞仪核心设施为他们的服务和帮助。

1.准备哺乳动物细胞，病毒生产和转导遗传条形码的<ol><li>板2.5×10 6的贴壁细胞在10cm的板（或约50-60％汇合）的前一天，以转染在Dulbecco改进的Eagle培养基（DMEM）和10％胎小牛血清（FCS）。逆转录病毒生产使用的包装细胞系的选择，如凤凰-GP（从加里·诺兰，斯坦福大学是一种礼物）。 注:该包装细胞系稳定表达的Gag和Pol蛋白对逆转录病毒颗粒形成中反式 。确保细胞是健康的，并附着于所述板中的单层，在转染之前。 </li><li> 24小时后，准备DNA转染混合物。 <ol><li>到125微升无血清的DMEM中加3微克水泡性口炎病毒包膜糖蛋白载体（PCI-VSVG）和3微克携带感兴趣的荧光蛋白的基因转移载体的构建。混合，并添加15微升聚乙（2毫克/毫升）。 </li><li>孵育混合物在室温下搅拌15分钟，并加入到细胞中逐滴。 注:聚乙加入到均匀的DNA混合物作为转染试剂为多聚物的形成。为了获得病毒颗粒携带不同的荧光蛋白标记物，以供选择的标记独立地进行每次转染。请记住，逆转录病毒转移载体是大约7 kb的长度，并可以不被打包如果超过10 kb的。 </li></ol></li><li>孵育板在37℃。为了增加病毒的生产场所板在30℃或32℃，而不是。 注:24小时后的转染介质可以用7ml的新鲜培养基代替，虽然这可能会改善细胞的健康也可能在上清液降低病毒载量。 </li><li> 48小时转染后收集含上清液的病毒颗粒和过滤器以0.45微米的聚四氟乙烯过滤器。使用新鲜收集病毒上清转导。 OTHerwise保持上清液以等分试样在-80℃下，并避免冻融。请记住，病毒滴度将减少可能高达50％，与每个冻融循环。 </li><li>优先选择24小时的板块细胞系传导，如果贴壁，或转导如果不贴前一刻。种子2.0×10 5〜3.0×10 5个贴壁细胞（这里咦7.5.1和HEK 293T）或5.0×10 5〜 1×10 6个非粘附细胞（此处SupT1）每孔在6孔板中。 注意:请确认校准单元的数目之前应当转导接种，特别对于贴壁细胞，所以细胞达到约60％的一汇合在转导的时间。 </li><li>添加5微克/ ml的凝聚胺（hexadimethrene溴化物）以接种细胞，并再加入之前收集的病毒上清以获得左右20-40％感染（这里是2毫升）中。 注:MOI或感染的比例大多是任意的，因为排序将允许放大任何subpopulat离子电池。显然，更高的速率感染的简化和加快了扩增的过程。 </li><li>在1500×g离心120分钟转导的细胞通过离心在悬挂桶转子在32℃。悬浮非粘附细胞用新鲜培养基之前孵化。对于贴壁细胞，更换新鲜培养基后24小时转导。 注:建议密封离心分离用封口膜的前板，以避免溢出。为了增加转基因条形码细胞多样性，转导的细胞与携带不同的荧光蛋白（来自步骤1.4）得到的病毒颗粒。当选择荧光标记物，要确保它们与仪器兼容，它们有区别的荧光参数。 </li></ol> 2.选择基因条码细胞和扩增<ol><li>通过流式细胞仪分析转导哺乳动物细胞至少72小时后传导到定量的百分比率转导。用可比较的非转导的细胞系作为阴性对照。 注意:作为排序将被用于纯化单个条形码的细胞群，高比例的速率初始转导，而不是必要的，应加快扩增的过程。 </li><li>利用细胞分选仪选择的，以获得表达目的蛋白质的细胞。 <ol><li>为了这个目的，确保栅极在右声道适当设定。 <ol><li>这样做使用相同的幼稚细胞系作为阴性对照，并设置参数/通道电压，使得负极人口低于10 3上的每个荧光轴上的值。确定选通荧光阳性如出现高于阴性对照为每个荧光通道的事件。 </li></ol></li><li>请注意，每个仪器可能会发生变化，并在正确的电压，用于确定选通应作相应的调整，使得必须在每一个记录阳性和阴性对照erimental设置。 <ol><li>荧光检测在本实验装置使用的FITC通道（由502长通滤波器进行三十零分之五百三十○带通滤波器），用于eGFP的，在PE通道（由556长通滤波器进行42分之585带通滤波器），用于TD番茄，和APC通道（二十〇分之六百六十〇带通滤波器），用于E2深红。 注:488 nm激光激发绿色荧光蛋白和TD番茄，而E2深红由633 nm激光激发。 </li></ol></li></ol></li><li>排序在15ml锥形管中，用1ml 100％的FCS。 注:排序荧光细胞群的过程不是强制作为一个可以直接进行单个克隆（见下文）的排序。排序根据所选荧光紧身人群，确保备份的人口为将来选择单个克隆。请记住，个体细胞是在时间难以生长，同时人口众多的细胞可以容易地冷冻，然后解冻，并放大在稍后阶段。 </li><li> SP在向下排序种群悬挂桶转子离心机以524克在32℃下5分钟以沉淀细胞。重新悬浮在1ml新鲜的培养基，并重新盘细胞，使细胞汇合是60％以下，以允许生长和分裂为至少两天。 <ol><li>例如，板的贴壁细胞在约3×10 5个细胞在2毫升每孔媒体在一个6孔板和2×10 6个细胞在2ml培养基中6厘米板。用DMEM培养贴壁细胞和RPMI对非贴壁细胞（或任何指明介质如果需要）。放置铺板细胞在培养箱中在37℃下进行数天，以允许扩增。 </li></ol></li></ol> 3.获取不同强度基因条码细胞克隆群<ol><li>从细胞中最初转导得到克隆群（步骤1.7）或从排序人口（步骤2.3）。 注意:这将确保在所有的细胞荧光蛋白的表达相同或相似的水平内的人口秒（一紧人口具有小的CV中的高达40％的平均荧光强度）。 <ol><li>为此目的，分类单个细胞到96孔板用自动细胞沉积单元（ACDU）。根据兴趣人群分类设置门。保证闸门都设置至少一个对数标度时，除了在不同的荧光强度选择细胞，以获得可区分种群。对于此处示出的过程中，使用相同的流式细胞仪实验设置为在步骤2.2中描述的。 </li></ol></li><li>扩增在培养箱单个克隆在37℃下为至少2周。通过扩增的过程中进行分析，在显微镜下的细胞，以确保不断增长的人口源自单个细胞而不是从多​​于一个。 注意:请记住，分拣机将置于每孔一个细胞的平均值，而一些孔可以不具有任何细胞可言，其他人可能有一个以上的。 <ol><li>为了确保婆pulation产生从一个小区只，是非常温和的与板和从未通过移液打扰的孔中。观察板块，深受很好，在显微镜下。 注意:虽然单个细胞有时可能难以确定，一旦他们开始除以他们将很容易辨认。 </li><li>要知道，往往细胞的丛&#39;沿边缘。抛弃那些井在多个无性系种群可以观察到，保留那些有一个严格的人口。 注意:最好是转移到那些较大的板块进行放大。 </li></ol></li><li>通过流式细胞仪分析这些筛选假定克隆群。比较它们的阴性对照使用相同流式细胞实验设置。 <ol><li>分析样品的唯一的百分比，并保持其余作为备份。选择的基础上平均强度和人口密封性最明显的不同人群。放大他们的需要或冻结股冷冻，用20％的甘油培养基在-​​80℃下的时间或液氮更长短时间。 注:请记住，"真正的"克隆群体应该有一个非常紧张的荧光图案。 </li></ol></li></ol> 4.确保复用功能的高通量筛选（HTS） <ol><li>根据需要，可以以多路复用格式进一步利用结合许多不同的克隆群。选择具有可区别的吸收/发射光谱和/或不同强度的克隆。如果一个96孔板格式的情况下，其适用于高温超导，种子50,000个细胞在200μl每孔。 注意:请注意，单元的数目只是一个估计，并且可以更改基于细胞类型以及它是否是附着或没有。 </li><li>使用流式细胞仪，以确保每个独立建立荧光细胞系可以相互区别开来的样品进行分析。在分析之前制备负和单一的颜色控制设置参数和补偿值。 注:设置的参数，以明确区分的混合人群，使他们进一步用在复用格式。 </li></ol> 5.适应基因条形码的细胞系选择的生物应用<ol><li>转导如斯托尔普等人，2013 59描述在这里建立遗传条形码的细胞系与包含所选择的测定元件的病毒颗粒。请记住，所选择的测定法应当占据额外的和可区别的荧光信道，因而它是被转移到相应的条形码的细胞系。 注:每个条码细胞系可承受不同的检测。 </li><li>排序基因萤光条形码细胞为那些含有测定的元素。 注:检测元件可改造耦合到标签或荧光蛋白（作为融合或通过内部核糖体进入位点）F或者简单的检测，通过免疫印迹，流式细胞仪和显微镜。在这个协议中使用的系统依赖于HA抗原表面表达单独或与另外的FLAG表位的表达。 <ol><li>含染色与医管局及APC-荧光抗体耦合的实验的克隆群。然后分析克隆群与抗FLAG和FITC缀合的抗体染色。 </li><li>为了跟踪背面的测定中，分析或"译码"在适当的信道的种群，其中所述不同的遗传条形码的细胞系能够被区分。这里，通过在对于TD番茄检测的体育频道分析解码所述衬底的性质。 </li></ol></li></ol>

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这里有两个既定的程序已合并;通过逆转录病毒技术和检测利用流式细胞荧光蛋白的基因工程。荧光蛋白的基于遗传条码用于生产独特的细胞系提供了一个强有力的和简单的方式用于多路复用应用。通过反转录病毒的技术产生的基因工程条码的细胞，最初是一个漫长的过程，但允许人们获得，一旦建立，电池材料的可靠和稳定的来源。此技术的性质一致地产生遗传改造的细胞系与成为自己真实区分biosignature稳定的荧光特性。 既粘附和非粘附细胞类型可利用条形码的荧光蛋白质，是容易区分基于其物理吸收/发射光谱。这些包括，但不限于蛋白质，如CFP，mCherry，E2深红和TD番茄。一旦基因条形码的其可区分荧光蛋白，它们可以被结合以产生细胞群的一个面板。重要的是，面板包含细胞群的确切基因型化妆，但只能通过它们的荧光特性差异。因此，这些面板都非常适合于复用的应用程序，大大增强了高通量的能力。 由于逆转录病毒颗粒进入宿主基因组中，再加上在MOI差异的不同插入的喜好，从进行特定荧光蛋白基因可变荧光强度被预期。遗传工程人口携带独特荧光基因的分析将因此检测的范围内，可以基于荧光强度来定义的表达谱。这个差异表达谱，可被利用来创建种群的光谱彼此不同。人们可以这样COMBINE选择的荧光强度的荧光标记物，以适应实验设计。这里，第4，第6，和第12群，其中包括的E2深红和TD番茄，有或没有的eGFP的两个6人口面板的矩阵，被示出。在理论上，这可以进一步扩展与eGFP的不同强度以及要创建的3种不同的荧光蛋白的人群;每一个具有与其关联的不同的强度。如前所述，应该考虑以确保分离选择的荧光蛋白时确实可以实现服用。选择的蛋白质应该有不同的吸收和发射光谱;然而，当两个光谱可区别的，但是类似的荧光蛋白时，补偿应适用。重要的是，适当的补偿，这允许对不同的荧光团或荧光蛋白之间发射/吸收光谱物理分离，完成与使用适当的单一颜色控制为通过流式细胞仪检测。 复用更少的荧光蛋白质，而且利用了各种强度的释放，可以再进一步开发用于感兴趣的生物应用的附加频道。这样可以提高的实验设置的灵活性并简化所使用的荧光标记物的组合的选择。例如，在图5所示的复用的测定法依赖于抗体染色，在这种情况下，联接到FITC。因为只有在PE通道由遗传条形码占据，人们可以利用FITC缀合的抗体，或与此相反，APC-耦合，可根据适当的仪器和/或抗体的可用性作出决定。 而在流动领域的技术成果术，显微镜或组合的方法是令人印象深刻的瓶颈似乎是可用的电池技术，生物应用和HTS。逆转录病毒技术术加上荧光蛋白质的扩大数目可以合并来回答这个查询。荧光遗传条码大幅促进复用，这反过来，满足了扩大的基于细胞的测定HTS能力的需求日益增加。遗传条码导致一个健壮的，具有成本效益，并且简单的方式来耦合的基于细胞的测定法与高通量方法用于生物应用和药物筛选。使用3人口面板执行一个而不是三个屏幕的力量有有益的成本和时间相关的影响。随着越来越多的多功能性在流式细胞仪，高内涵成像和流式细胞仪耦合显微镜，荧光遗传条形码将是试验发展持续可靠的工具。

技术，如荧光光谱，荧光显微镜，流式细胞仪，全部依靠荧光，属性广泛利用生物化学，生物医药，化工等应用。荧光，无论本征或经标记，已经开发用于蛋白质表达的模式和分布，细胞命运，蛋白质相互作用和生物功能1-9的分析，以及通过对生物分子的相互作用和构象变化的荧光检测/荧光共振能量转移10-13。由于Aequorea victoria的绿色荧光蛋白（GFP）14的分离，从其他腔肠动物，特别是珊瑚附加天然存在的荧光蛋白质的发现，已经在很大程度上增加了现有的荧光蛋白质与可区别的激发/发射光谱的数目。这些，在他们的基因15-19引入突变在一起，甲肝Ë进一步扩大的可能性，获得提供给科学家们利用显微镜，流式细胞仪等荧光技术用于他们的研究荧光蛋白的真实调色板。 与此同时，虽然独立地，逆转录病毒技术的发展已大大促进了异位遗传信息的稳定表达在哺乳动物细胞20-23。因此，这是不奇怪，这种技术已被用来传输的荧光蛋白的基因导入细胞类型和组织24-28的广泛号码或用于生产转基因动物的29-31。以下逆转录病毒的性质，异位荧光蛋白的遗传信息的小区32的基因组中导入和细胞就荧光`为ever&#39;。该属性允许内细胞群的单个细胞的细胞命运的跟踪，或。现在荧光细胞也由此阿奎红其自身biosignature并且可以被定义为条形码的。其独特的biosignature来自其他小区的识别它，并且重要的是，从细胞中遗传操作以表达不同的荧光蛋白与可区分的吸收/发射光谱区分它。生物应用，例如重编程因子的跟踪朝向多潜能33的亚核因素的分析为核仁定位34的阐明，荧光报告质粒的构建转录研究35或神经元的神经元网络体系结构36的研究中的遗传标记，是那些利用不同的荧光蛋白基因相同的实验设置的许多只是四个例子。 流式细胞术已被广泛用于生物过程在单细胞水平，如基因表达，细胞周期，细胞凋亡的分析，并通过磷的信令荧光蛋白基因在哺乳动物细胞ATION 37-43 .The稳定表达进一步增强了流式细胞仪的细胞分析38,44和配体-受体相互作用45的效用。增强的功能已经允许流式细胞成为高通量和高含量筛选46一种广泛使用的方法。尽管可以耦合板读数器系统，成像和流式细胞术荧光计和机器人技术现在扩大数量，似乎有一个缺少的实验设计，可利用和适合这些增强技术能力。 有大幅度需要复用的应用进一步提升的高吞吐能力快速，可靠，简单而强大的基于细胞的方法。这是在药物发现领域尤其如此，其中以多路复用格式的工程基于细胞的测定可增强高通量筛选39,47-50电源</sup&gt;。复用的，因为它允许同时分析一个样本，进一步增强了高吞吐量能力51-54。荧光遗传条码不仅允许优雅复用，而且，一旦改造，规避了耗时的协议的需要，降低了成本伴随抗体，珠子和污渍39,52,55，并且可以减少所需的高屏幕数吞吐量的应用。我们最近已描述的技术如何逆转录病毒可以增强通过荧光遗传条码多路复用生物应用，通过表达以前开发并监控HIV-1蛋白酶活性的56,57具有不同的临床流行变体58的测定法。该方法是在一个更具描述性的方式解释着眼于如何选择和基因扩增荧光条码细胞如何产生克隆群体的表达板不同的荧光蛋白和/或不同的荧光intensitIES。细胞群区分基于其荧光特性的面板增强多路复用能力，它可以被进一步利用结合基于细胞的试验是对付不同的生物的问题。该协议还描述了如何对工程师条形码细胞轴承先前在实验室开发的基于细胞的测定中的一个的板，如实施例59。这个协议是因此不意图以显示既定的逆转录病毒/慢病毒技术进行基因转移，荧光蛋白的价值或流式细胞仪60,48的应用程序，而是以显示结合三个用于多路复用应用的增强能力。

<table><tbody><tr><td>10 ml syringes</td><td>BD</td><td>309604</td><td>used for filtering the virus</td></tr><tr><td>0.45 &amp;micro;m ploytetrafluoroethylene filter</td><td>Pall Corporation</td><td>4219</td><td>used for filtering the virus</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Corning</td><td>45000-304</td><td>cell growth media for HEK 293T cells</td></tr><tr><td>PEI (Polyethylenimine)</td><td>poly sciences</td><td>23966-2</td><td>2 mg/ml concentration used</td></tr><tr><td>Hanging bucket centrifuge (refrigerated)</td><td>Eppendorf</td><td>5805 000.017</td><td>used for spin infection</td></tr><tr><td>PBS (phosphate buffered saline)</td><td>Corning</td><td>21-040-CV</td><td>used for washing of cells</td></tr><tr><td>Polybrene (hexadimethreen bromide)</td><td>Sigma-Aldrich</td><td>107689</td><td>Used to increase viral infection efficiency. Used at a 5 &amp;micro;g/ml concentration.</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>instrument used for sorting cell populations</td></tr><tr><td>FACSCanto</td><td>BD Biosciences</td><td></td><td>instrument used for cell analysis</td></tr><tr><td>Phoenix-GP</td><td>Gift from Gary Nolan</td><td></td><td>cell line used to produced retroviral particles</td></tr><tr><td>Fetal calf serum</td><td>Mediatech</td><td>MT35015CV</td><td>used for cell growth and sorting</td></tr><tr><td>SupT1 cells</td><td>ATCC</td><td>CRL-1942</td><td>Human T lymphoblasts</td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td>Human Embryonic Kidney cells that also contain the SV40 large T-antigen</td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CV</td><td>cell growth media for SupT1 cells</td></tr></tbody></table>

genetic barcoding with fluorescent proteins for multiplexed applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

复用荧光基因条形码的细胞的生物应用的目的，才能达到一次个别克隆群已经产生。当条形码的人群与最低频谱重叠清晰鲜明的特色荧光复用是最有效的。在图1与哺乳动物SupT1细胞的克隆群所示的例子中示出了条形码的细胞mCherry和氰基荧光蛋白（CFP）可以容易地同时进行分析，而不会失去它们各自的荧光特性。此矩阵从而体现荧光基因条形码的单元的面板是用于在又一个附加的可利用信道复用的生物应用的读出使用。为了获得这样的面板的是要记住逆转录病毒技术的性质，这将导致种群内的荧光强度由于插入EF变量范围是非常重要的fects和/或MOI; 图2示出了哺乳动物细胞用含有病毒颗粒或者TD番茄或E2深红导致细胞表达的任何一个所述的荧光蛋白质或两者在宽范围的强度（左面板）的初始转导。选择和如由门控箱（中间面板）的单细胞到96孔板的排序，允许人们获得如下扩展（右面板）紧克隆群。甲紧人口应该被定义为具有小的CV其可根据细胞类型的范围，通常30-40％的平均荧光强度。 图2还示出，为了进一步提高基因条形码种群的数目仅为2荧光的基质蛋白质，还可以利用荧光强度。通常，在人口之间的平均荧光强度1日志偏差适于彼此以下实现适当的分离和分拣amplifica化。 TD番茄被选为在所示的例子来说明该特征，其中不同的强度，中，高的两个群体，均在一个单一的强度而获得，用于多路复用与E2的深红。 复用是一种强大的工具，以促进许多样品的分析的同时，并用于解码掩蔽种群的能力。增强多路复用可以与目前的第三荧光蛋白如绿色荧光蛋白来实现，只要光谱性质不相互干扰。在图3中所示的实验程序，用TD番茄和E2深红得到6个种群的面板被利用来增加复用的eGFP。逆转录病毒的技术被用来eGFP的转移到在所述矩阵中的一个表示的种群。当在eGFP的信道观察到的，非绿色幼稚六人口矩阵（左上面板在图3中）是从的eGFP转导的区分人口矩阵（左下面板在图3中）。重要的是，该板可以通过原有的遗传条形码占据了渠道分析（TD番茄和E2深红）。而无法 ​​区分在这些通道中（比较人口1-6与种群7-12在板中间， 图3）中所示的直方图，个体群体可以在eGFP的信道进行分析，并解码或跟踪背部，（右图图3）。重复转导，选择，排序，和放大的过程中，以未占用信道的优势之后，如果合适的补偿不施加到调整为可能的光谱重叠是无用的。为了证明这一点，当群体1，2，3（非绿色）和7，8，9（绿色）中的eGFP分析和TD番茄通道，群7和8是难以区分（ 图4中左面板） 。因此，有必要选择幼稚细胞（群1），为负CONTROL使得仪器的参数进行设置。在分析的任何基质，尤其是与荧光团的光谱重叠，当务之急是单一颜色控制用于​​确定正确的补偿值。在图1的分析4个群体2或3，和7达到这个目的，从而允许更好地确定种群，是真正的双阳性（人口8和9）。 遗传条形码的细胞与荧光标记物保持其自己的身份通过biosignature所定义，当在右荧光通道与右仪器进行分析。然而，biosignature变成只是一个额外的个人财产，除非开发用于生物应用。为了证明荧光遗传条码的功率复用，我们决定介绍我们以前开发试验用于药物发现中的一个到一些荧光条形码哺乳动物细胞系。通过这样做，fluoresc耳鼻喉科遗传条形码被利用来实现三个试验中的一个样本。在此过程中含有三种公认的病毒基板蛋白酶解1支架蛋白转移到经遗传条形码的细胞。在测定中，切割是通过在细胞表面上（ 图5B）的FLAG表位的丧失揭示。与此相反，FLAG表面染色表示缺乏卵裂图5示出了三个不同的基板上的分析结果。 HIV-1包膜野生型（环境质量），HIV-1包膜突变（环境MUT）和登革热病毒（DENV）的prM。他们每个人都被引入到1 3的条形码的细胞系，幼稚，和2个其他利用TD番茄不同强度（中高; 图5A）。染色FLAG表面表达揭示了其中衬底是基于各自的条形码裂解。在该例子中，只有HIV的Env MUT保留了FLAG标记（阳性染色以下），如在绿色-FI看出TC通道（ 图5C，右侧底部面板）。因此，可以进行独立于原始条形码分析的分析，并得到进一步利用来解码和追溯的不同群体。多路复用的这种方法因而依赖于保留给感兴趣的生物读出的附加信道。 <img alt="图1" src="/files/ftp_upload/52452/52452fig1highres.jpg" width="700" /> 图1:条码，用于多路复用各个群体使用不同的荧光蛋白如mCherry，CFP或二者（左面板）基因条形码的可混合，分析和解码（右图）在它们各自的通道，通过流式细胞仪。从Smurthwaite，C。等2014 58改编。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52452/52452fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO:保持together.within页="总是"&gt; <img alt="图2" src="/files/ftp_upload/52452/52452fig2highres.jpg" width="700" /> 图2:分拣和条形码细胞扩增的哺乳动物细胞进行了分析48小时以下转导与含有任一TD番茄或E2-深红（左图）的病毒颗粒。盖茨然后进行排序，包括细胞表达TD番茄不同强度设定，与输入/输出E2-深红（中间面板）。从各种各样的克隆扩增和重新分析，以产生6个区分人群（右图）的矩阵。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52452/52452fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52452/52452fig3highres.jpg" width="700" /> 图3:解码揭示掩蔽种群的基质在图2（TD番茄和/或E2-深红）中获得6个种群可以进一步工程化以表达一个额外的荧光蛋白，如绿色荧光蛋白。 （A） 的原始矩阵，当信道的eGFP（左图）分析是阴性的6个种群彼此区分。每六个种群可以在eGFP的信道被独立地分析所示的直方图（右图）。 （ 二）同一矩阵，现在也表达绿色荧光蛋白，分析在A，揭示目前他们的绿色荧光特征。从Smurthwaite，C。等2014 58改编。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52452/52452fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52452/52452fig4highres.jpg" width="500" /> 图4:补偿确保正确的分离部分人群的最初选择基于TD番茄和/或EGFP表达（人口7和8）不能当一起分析正常分离（左图），除非这些渠道给予相应的补偿调整（右。面板）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52452/52452fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52452/52452fig5highres.jpg" width="700" /> 图5:选择条码细胞进一步适应所选检测的（A）选择和适应的选择检测。种群基因条形码的与TD番茄不同强度（左上面板）用于生物应用。每个种群的进一步工程化以包含监视裂​​解的测定的，但不同的衬底中的每一个（右上面板）。 （B）描写了测定。积极FLAG染色表明缺乏裂解，而负FLAG染色表明乳沟。测定的（C）的分析如下FLAG染色。基于所述TD番茄条形码但无法区分在FITC通道（左小图）的混合种群是可区别的。当与FLAG-FITC染色的抗体，只有一个群体是积极的FLAG。解码显示，基于遗传条形码，这个群体蕴藏着HIV包膜MUT基板（右图）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52452/52452fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications at JoVE.com

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

genetic-barcoding-with-fluorescent-proteins-for-multiplexed

Universidad San Sebastian

Research

JoVE Journal

Biology

9.2K 次观看.  San Diego State University. Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications. 

视频: Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

科研

免疫与感染

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

遗传学

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

癌症研究

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

发育生物学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

生物学

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

采用多转染的细胞状态识别电路的快速开发

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...

生物工程

BEST: Barcode Enabled Sequencing of Tetrads

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Barcode Enabled Sequencing of Tetrads (BEST) replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by fluorescence-activated cell sorting onto agar plates, separates the spores by agitation with glass beads, and determines which randomly arrayed colonies were derived from the same original tetrad using molecular barcodes.

最佳：条码启用四分体测序

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

神经科学

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...