遗传条形码与荧光蛋白的复用的应用程序
Summary
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.
Abstract
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 ´indefinitely´. 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.
Introduction
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'。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>。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。
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| 10mL syringes | BD | 309604 | used for filtering the virus |
| 0.45µm plytetrafluoroethylen filter | pall corporation | 4219 | used for filtering the virus |
| DMEM (Dulbecco's Modified Eagle Medium) | Corning | 45000-304 | cell growth media for HEK 293T cells |
| PEI (Polyethylenimine) | poly sciences | 23966-2 | 2mg/mL concentration used |
| Hanging bucket centrifuge (refrigerated) | Eppendorf | 5805 000.017 | used for spin infection |
| PBS (phosphate buffered saline) | Corning | 21-040-CV | used for washing of cells |
| Polybrene (hexadimethreen bromide) | Sigma-Aldrich | 107689 | Used to increase viral infection efficiency. Used at a 5µg/mL concentration. |
| FACSAria | BD Biosciences | instrument used for sorting cell populations | |
| FACSCanto | BD Biosciences | instrument used for cell analysis | |
| Phoenix-GP | Gift from Gary Nolan | cell line used to produced retroviral particles | |
| Fetal calf serum | Mediatech | MT35015CV | used for cell growth and sorting |
| SupT1 cells | ATCC | CRL-1942 | Human T lymphoblasts |
| HEK 293T cells | ATCC | CRL-11268 | Human Embryonic Kidney cells that also contain the SV40 large T-antigen |
| RPMI 1640 | Corning | 10-040-CV | cell growth media for SupT1 cells |
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