使用提顿体系的研究斑马鱼再生的分子机制
Summary
Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.
Abstract
The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta–catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.
Introduction
斑马鱼是一种行之有效的脊椎动物模式生物研究的发展，生理学，疾病和再生的许多方面。随着越来越多地采用斑马鱼作为模型用于后胚胎的生物过程，实验工具为诱导型，组织特异性操纵基因表达或信号转导途径已经变得越来越重要。具体地讲，研究成器官和附属物再生在成年斑马鱼都遭受缺乏工具的过程中，这些再生过程信号通路的时空要求清扫。
目前，三种不同的系统已被用于实现在再生成年斑马鱼的内脏有条件的，组织特异性的基因表达：在的Cre-lox系统中，使用转座子介导的体细胞的转基因热休克诱导转基因拼接表达，并且顿系统1 -3。堤是指四环素续的变体轧制转录激活系统，其中表达在抗生素四环素或其衍生物， 例如多西环素存在下激活。在的Cre-lox系统，因为它迄今已在成年鱼使用的，依赖于他莫昔芬控制Cre重组酶（CreERT2），其表达在空间上受限于组织特异性调控元件。 Cre的驱动除去一个STOP盒的促进的兴趣的启动子应是活性在所有细胞类型1驱动的基因的表达。转座子介导的创作mosaically表达的转基因体细胞提供了在单个细胞系诱导转基因表达系统。注射斑马鱼的胚胎与TOL2座子携带目的基因下的热休克启动子的结果在嵌合个体通常携带外源基因仅在再生器官2的离散细胞谱系的转录控制。虽然这两个系统允许有条件的组织SPEcific基因表达方法，将Cre-lox系统是不可逆的，并且使用转座子为基础的克隆标记策略从它的随机性质受到影响。因此，我们和其他人已经最近适于转基因TETON系统用于在斑马鱼中，这有利于在时间上和空间上控制基因的表达是在附加的可调和可逆3-5。
此处所用的提顿系统包括转基因驱动线（TetActivator），其中多西环素（DOX）诱导性转录激活（改进反向四环素反式激活，irtTA，短TETA）是根据组织特异性基因调控序列的控制。其次，它要求的转基因应答线（TetResponder），该港口的在转录控制下的四环素操作员感兴趣的基因（四环素响应元件; TetRE）（ 图1A）。因此，使用的TetActivator和TetResponder线路特定组合允许有条件组织特定基因表达的FIC操纵。
最近，我们利用了系统顿探测的Wnt /β-catenin信号通路的成人斑马鱼再生尾鳍3组织特异性的功能。在这里介绍的协议中，我们描述了用于在斑马鱼中的建立的顿系统和使用，特别是用于鳍再生的研究工作流。这包括如何生成稳定的转基因TetActivator和TetResponder线的详细说明和协议，用于在胚胎和成年斑马鱼转基因诱导。此外，我们描述了在鳍再生技术验证组织特异性基因表达，包括一个协议的成年斑马鱼翅片冷冻切片的制备方法。此外，我们将讨论考虑的TetActivator转基因的表达TetResponder的设计，的转基因方法的选择和检测。因此，该协议的总体目标是作为一个蓝图设置式的官能顿系统在斑马鱼实现有条件组织特异性基因的表达，这可以适用于任何感兴趣的组织。
我们已经创建了一个TetActivator向量允许I-SceI的或TOL2介导的一代使用短基因的调控序列稳定TetActivator线（增强片段; Weidinger实验室质粒数据库中没有1247; 图1B）。此构建体含有一个TetActivator盒，其组成稠合与单纯疱疹病毒VP16的激活结构域衍生3F [irtTAM2（3F）]反向的Tet阻抑物结构域的M2突变体变体。所述TetActivator（TETA）的表达可以容易地监测，因为它是共表达与来自相同的开放阅读框架的荧光团AmCyan;一个P2A肽介导核糖体跳跃，这将导致生产TETA和AmCyan截至1独立的蛋白质：1的比例5,6。该构建体也包含一个poylinker 5'T形etActivator盒，以便于插入的兴趣使用常规的克隆方法的基因组调节序列。
此外，我们已经创建了一个构建体由上述TetActivator盒外加一个卡那霉素选择盒（Weidinger实验室质粒数据库没有1180; 图1C）含有大量的基因组区域，可以重组到细菌人工染色体（BAC）（通常成基因的表达模式是由转基因来模拟）的起始密码子。这两种结构都可以从应要求Weidinger实验室。
Protocol
Representative Results
Discussion
成年斑马鱼有一个惊人的能力，成功地再生许多内部器官和附属物。深入了解涉及的分子和细胞机制的需要的基因功能和信号通路组织特异性分析。朝此，特顿系统提供了在胚胎和成年斑马鱼spatiotemporally控制基因表达的有效工具。特顿体系结构和方法论在这个手稿中描述已成功应用于最近我们实验室3的研究。建立系统顿时下列几个问题应考虑：
TetActivator转基因?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢克里斯塔·哈泽，多丽丝·韦伯和布里吉特科特技术援助。在Weidinger实验室工作是由德意志研究联合会WE 4223 / 3-1的补助，我们4223 / 4-1和德意志GESELLSCHAFT献给Kardiologie通过奥斯卡 – 拉普 – Stipendium和克劳斯 – 乔治·UND-Sigrid- Hengstberger-Forschungsstipendium。
Materials
| Breeding boxes | Aqua Schwarz | AquaBox 1 | |
| Compound fluorescent microscope | e.g. Leica, Zeiss | varies with the manufacturer | to image fluorescent tissue sections |
| Confocal microscope | e.g. Leica, Zeiss | varies with the manufacturer | to image fluorescent tissue sections |
| Cryostat | e.g. Leica, Thermo-Scientific | varies with the manufacturer | for cryosectioning |
| 4’, 6- diamidino-2-phenylinodole (Dapi) | Sigma-Aldrich | D9542 | use 1/5000 in PBS for visualization of nuclei |
| Doxycycline | Sigma-Aldrich | D9891 | prepare stocks in 50% EtOH at 50 mg/ml (97 mM) for TetResponder induction |
| E3 embryo medium | 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2· 2 H2O, 0.33 mM MgSO4·7 H2O, 0.2 ‰ (w/v) methylene blue, pH 6.5 for embryo/larvae husbandry |
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| Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | 4 % (w/v) paraformaldehyde in PBS, pH 7.5 for fixation |
| 1x Phosphat-buffer saline (PBS) | 1.7 mM KH2PO4, 5.2 mM Na2HPO4, 150 mM NaCl, pH 7.5 | ||
| 1x Phosphat-buffer saline + Tween 20 (PBT) | 1x PBS with 0.1 % Tween 20 | ||
| Superfrost Ultra Plus adhesion microscope slides | Thermo Scientific | 1014356190 | for collection of tissue sections |
| Stereo fluorescent microscope | e.g. Leica, Zeiss | varies with the manufacturer | for fluorescence-based genotyping |
| Thermocycler | e.g. Biorad, Applied Biosystems | varies with the manufacturer | for PCR-based genotyping |
| Tissue freezing medium (TFM) | Triangel Biomedical Sciences | TFM-C | for embedding of tissue samples |
| Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222) | Sigma-Aldrich | E10521 | for anesthesia use at 1 mg/ml in E3 embryo medium |
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