斑马鱼 Tol2 系统：一种模块化且灵活的基于网关的转基因方法
Summary
这项工作描述了模块化Tol2转基因系统的协议，这是一种基于网关的克隆方法，用于创建转基因构建体并将其注入斑马鱼胚胎。
Abstract
胎儿酒精谱系障碍 （FASD） 的特征是一组高度可变的结构缺陷和认知障碍，这些缺陷和认知障碍是由于产前乙醇暴露引起的。由于胎儿酒精谱系障碍的复杂病理学，动物模型已被证明对我们目前对乙醇诱导的发育缺陷的理解至关重要。斑马鱼已被证明是检查乙醇诱导的发育缺陷的有力模型，因为斑马鱼和人类之间的遗传和发育高度保守。作为一种模型系统，斑马鱼具有许多属性，使其成为发育研究的理想选择，包括大量遗传可处理和半透明的外部受精胚胎。这使研究人员能够在多种遗传环境中精确控制乙醇暴露的时间和剂量。斑马鱼中可用的一个重要遗传工具是转基因。然而，生成转基因构建体和建立转基因系可能是复杂和困难的。为了解决这个问题，斑马鱼研究人员建立了基于转座子的Tol2转基因系统。该模块化系统使用多位点网关克隆方法快速组装完整的基于Tol2转座子的转基因构建体。在这里，我们描述了灵活的Tol2系统工具箱和用于生成可用于斑马鱼转基因的转基因构建体及其在乙醇研究中的使用的方案。
Introduction
产前乙醇暴露会导致连续的结构缺陷和认知障碍，称为胎儿酒精谱系障碍（FASD）1，2，3，4。多种因素之间的复杂关系使得研究和理解人类胎儿酒精谱系障碍的病因具有挑战性。为了解决这一挑战，已经使用了各种各样的动物模型。事实证明，这些模型中可用的生物学和实验工具对于发展我们对乙醇致畸性的机理基础的理解至关重要，并且这些模型系统的结果与人类乙醇研究中发现的结果非常一致5，6。其中，斑马鱼已成为研究乙醇致畸7，8的有力模型，部分原因是它们的外部受精，高繁殖力，遗传可移植性和半透明胚胎。这些优势相结合，使斑马鱼成为使用转基因斑马鱼系对胎儿酒精谱系进行实时实时成像研究的理想选择。
转基因斑马鱼已被广泛用于研究胚胎发育的多个方面9。然而，创建转基因构建体和随后的转基因品系可能非常困难。标准转基因需要一个活性启动子元件来驱动转基因和一个多聚A信号或“尾巴”，所有这些都在一个稳定的细菌载体中，用于一般载体维护。传统一代的多组分转基因构建体需要多个耗时的亚克隆步骤10。基于聚合酶链反应的方法，如吉布森组装，可以规避与亚克隆相关的一些问题。然而，必须设计和测试独特的引物，以产生每个独特的转基因构建体10。除了转基因构建之外，基因组整合、种系传播和筛选适当的转基因整合也很困难。在这里，我们描述了使用基于转座子的Tol2转基因系统（Tol2Kit）的协议10，11。该模块化系统使用多位点网关克隆，从不断扩展的“入口”和“目标”载体库中快速生成多个转基因构建体。集成的Tol2转座元件大大提高了转基因速率，允许多个转基因的快速构建和基因组整合。使用该系统，我们展示了如何使用内胚层转基因斑马鱼系的产生来研究胎儿酒精谱系背后的组织特异性结构缺陷。最终，在该协议中，我们表明转基因构建体的模块化设置和构建将极大地帮助基于斑马鱼的FASD研究。
Protocol
该程序中使用的所有斑马鱼胚胎均按照既定的IACUC协议12进行饲养和繁殖。这些协议得到了路易斯维尔大学的批准。 注意：本研究使用了野生型斑马鱼品系AB和 bmp4st72;smad5b1100 双突变株系。该程序中使用的所有水都是无菌反渗透水。在激光扫描共聚焦显微镜下拍摄共聚焦图像。内胚层测量是使用ImageJ中的测量工具进行的。所有统计分析?…
Representative Results
为了生成转基因构建体，我们使用了Tol2转基因系统。三种进入载体，包括包含基因启动子/增强子元件的p5E，保存由启动子/增强子元件表达的基因的pME和至少包含polyA尾巴的p3E，用于通过多位点网关LR克隆 生成 转基因构建体。目标载体pDest为斑马鱼胚胎中转基因构建体的基因组插入提供Tol2重复序列，并包含细菌生长的所有基本遗传信息（图1A）。出于本文的目的，我?…
Discussion
斑马鱼非常适合研究乙醇暴露对发育和疾病状态的影响7，8。斑马鱼产生大量半透明、外部受精、可遗传处理的胚胎，这允许在多种环境背景下同时对几种转基因标记的组织和细胞类型进行活体成像19，20。这些优势，加上对人类强大的发育遗传保护，使斑马鱼成为对乙醇7，<sup class="…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本文中介绍的研究得到了美国国立卫生研究院/国家酒精滥用研究所（NIH/NIAAA）R00AA023560对C.B.L的资助。
Materials
| Addgene Tol2 toolbox | https://www.addgene.org/kits/cole-tol2-neuro-toolbox/ | ||
| Air | Provided directly by the university | ||
| Ampicillin | Fisher Scientific | BP1760 | |
| Analytical Balance | VWR | 10204-962 | |
| Borosil 1.0 mm OD x 0.75 mm ID Capillary | FHC | 30-30-0 | |
| Calcium Chloride | VWR | 97062-590 | |
| Chloramphenicol | BioVision | 2486 | |
| EDTA | Fisher Scientific | BP118-500 | |
| Fluorescent Dissecting Microscope | Olympus | SZX16 | |
| Kanamycin | Fisher Scientific | BP906 | |
| Laser Scanning Confocal Microscope | Olympus | Fluoview FV1000 | |
| Lawsone Lab Donor Plasmid Prep | https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols/ | ||
| LB Agar | Fisher Scientific | BP9724 | |
| LB Broth | Fisher Scientific | BP1426 | |
| Low-EEO/Multi-Purpose/Molecular Biology Grade Agarose | Fisher Scientific | BP160-500 | |
| LR Clonase II Plus Enzyme | Fisher Scientific | 12538200 | |
| Magnesium Sulfate (Heptahydrate) | Fisher Scientific | M63-500 | |
| Micro Pipette holder | Applied Scientific Instrumentation | MIMPH-M-PIP | |
| Microcentrifuge tube 0.5 mL | VWR | 10025-724 | |
| Microcentrifuge tube 1.5 mL | VWR | 10025-716 | |
| Micromanipulator | Applied Scientific Instrumentation | MM33 | |
| Micropipette tips 10 μL | Fisher Scientific | 13611106 | |
| Micropipette tips 1000 μL | Fisher Scientific | 13611127 | |
| Micropipette tips 200 μL | Fisher Scientific | 13611112 | |
| mMESSAGE mMACHINE SP6 Transcription Kit | Fisher Scientific | AM1340 | |
| Mosimann Lab Tol2 Calculation Worksheet | https://www.protocols.io/view/multisite-gateway-calculations-excel-spreadsheet-8epv599p4g1b/v1 | ||
| NanoDrop Spectrophotometer | NanoDrop | ND-1000 | |
| NcoI | NEB | R0189S | |
| NotI | NEB | R0189S | |
| Petri dishes 100 mm | Fisher Scientific | FB012924 | |
| Phenol Red sodium salt | Sigma Aldrich | P4758-5G | |
| Pipetman L p1000L Micropipette | Gilson | FA10006M | |
| Pipetman L p200L Micropipette | Gilson | FA10005M | |
| Pipetman L p2L Micropipette | Gilson | FA10001M | |
| Potassium Chloride | Fisher Scientific | P217-500 | |
| Potassium Phosphate (Dibasic) | VWR | BDH9266-500G | |
| Pressure Injector | Applied Scientific Instrumentation | MPPI-3 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | |
| Sodium Bicarbonate | VWR | BDH9280-500G | |
| Sodium Chloride | Fisher Scientific | S271-500 | |
| Sodium Phosphate (Dibasic) | Fisher Scientific | S374-500 | |
| Stericup .22 µm vacuum filtration system | Millipore | SCGPU11RE | |
| Tol2 Wiki Page | http://tol2kit.genetics.utah.edu/index.php/Main_Page | ||
| Top10 Chemically Competent E. coli | Fisher Scientific | C404010 | |
| Vertical Pipetter Puller | David Kopf Instruments | 720 | |
| Zebrafish microinjection mold | Adaptive Science Tools | i34 |
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Cite This Article
Klem, J. R., Gray, R., Lovely, C. B. The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach. J. Vis. Exp. (189), e64679, doi:10.3791/64679 (2022).