Name: the zebrafish tol2 system: a modular and flexible gateway-based transgenesis approach
Uploaded: 2022-11-30T14:50:00
Description: Watch this Scientific Journal Video about The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach at JoVE.com

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。

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?...

<ol>
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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="...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Addgene Tol2 toolbox</td>
			<td>https://www.addgene.org/kits/cole-tol2-neuro-toolbox/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Air</td>
			<td></td>
			<td></td>
			<td>Provided directly by the university</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>10204-962</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosil 1.0 mm OD x 0.75 mm ID Capillary</td>
			<td>FHC</td>
			<td>30-30-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>BioVision</td>
			<td>2486</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP118-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lawsone Lab Donor Plasmid Prep</td>
			<td>https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP9724</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-EEO/Multi-Purpose/Molecular Biology Grade Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Clonase II Plus Enzyme</td>
			<td>Fisher Scientific</td>
			<td>12538200</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (Heptahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Pipette holder</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MIMPH-M-PIP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 0.5 mL&#160;</td>
			<td>VWR</td>
			<td>10025-724</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL&#160;</td>
			<td>VWR</td>
			<td>10025-716</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 10 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 1000 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611127</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 200 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611112</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Fisher Scientific</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosimann Lab Tol2 Calculation Worksheet</td>
			<td>https://www.protocols.io/view/multisite-gateway-calculations-excel-spreadsheet-8epv599p4g1b/v1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>NanoDrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>NEB</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>NEB</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 100 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>P4758-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p1000L Micropipette</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p200L Micropipette</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p2L Micropipette</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Dibasic)</td>
			<td>VWR</td>
			<td>BDH9266-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Injector</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>VWR</td>
			<td>BDH9280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup .22 &#181;m vacuum filtration system&#160;</td>
			<td>Millipore</td>
			<td>SCGPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tol2 Wiki Page</td>
			<td>http://tol2kit.genetics.utah.edu/index.php/Main_Page</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Top10 Chemically Competent E. coli</td>
			<td>Fisher Scientific</td>
			<td>C404010</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Pipetter Puller</td>
			<td>David Kopf Instruments</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>i34</td>
			<td></td>
		</tr>
	</tbody>
</table>

the zebrafish tol2 system: a modular and flexible gateway-based transgenesis approach

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。

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?...

Watch this Scientific Journal Video about The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach at JoVE.com

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

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。

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

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

转基因斑马鱼已被证明对我们理解胚胎发育至关重要。Tol2系统是一种多功能工具，使研究人员能够快速系统地生成多种转基因斑马鱼用于FASD研究。模块化设计和易于生成新的试剂盒组件使 Tol2 系统成为生成新转基因试剂盒的多功能工具，无需重新设计试剂盒的任何组件。我对首次使用这项技术的研究人员的建议是进行几次试运行，以训练生成和注入转基因构建体的精度。首先，通过在 500 μL 微量离心管中合并 10 μL 转座酶质粒、1.5 μL 限制性核酸内切酶反应缓冲液和 0.3 μL nought 一种核酸内切酶，将含有转座酶的 Tol2 试剂盒质粒与 1 个限制性核酸内切酶线性化。用不含RNAse的水填充反应至20微升。在 37 摄氏度下混合并消化过夜。第二天，通过向反应混合物中加入一微升0.5摩尔EDTA，两微升五摩尔乙酸铵和80微升100%乙醇来停止消化。混合并在零下 20 摄氏度下冷却过夜。第二天，离心并吸出上清液，然后将DNA沉淀重悬于无RNA酶的水中。然后通过运行两微升样品在荧光计上确定线性质粒浓度（以纳克/微升为单位）。通过将 10 微升 2X NTP/CAP、两微升 10X 反应缓冲液、0.1 至 1 微克线性 DNA 模板与 500 微升管中的两微升 SP6 酶混合物混合来设置 SP6 mRNA 生产。用不含RNA酶的水填充反应至20微升，并在37摄氏度下孵育之前混合。两小时后，加入一微升DNA酶，充分混合，再孵育15分钟。通过加入30微升氯化锂沉淀溶液停止反应。混合并在零下 20 摄氏度下冷却过夜。第二天，离心溶液以沉淀mRNA并吸出上清液，然后用一毫升80%乙醇洗涤沉淀。风干沉淀并将其重悬于20微升无RNA酶的水中。使用荧光计测定 mRNA 浓度，然后每管等分 100 纳克 mRNA 供一次性使用，并储存在零下 80 摄氏度。为了进行反应，收集 P5E、PME 和 P3E 各 10 飞摩尔和目标载体 PDEST 的 20 飞摩尔。从质粒源鉴定所有四种载体和碱基对的大小。在确定所有四种载体的浓度后，使用DNA重量为660克/摩尔以及质粒大小和碱基对，计算进入载体达到10飞摩尔或目标载体达到20飞摩尔所需的质粒总纳克。接下来，使用手稿中描述的组件生成构造 sox17：EGFP-CAAX。使用质粒浓度，计算达到10飞摩尔或20飞摩尔所需的每个质粒的总微升，然后将其除以2以用于5微升半LR反应。生成 10 飞摩尔的 P5E sox17、PME EGFP-CAAX、P3E Poly（A） 和 20 飞摩尔的目标载体。通过在 500 微升管中合并 P5E、PME、P3E 和 PDEST 的体积来建立 5 微升半 LR 反应。然后加入无菌水，使体积为四微升。涡旋LR酶混合物两次，每次一分钟，然后加入一微升反应中，并在25摄氏度下孵育之前彻底混合。第二天，通过加入0.5微升蛋白酶K停止LR反应，在37摄氏度下孵育10分钟，然后冷却至室温。接下来，通过添加质粒并使细胞在冰上放置 25 到 30 分钟，将三到四微升质粒转化为解冻的化学感受态细胞。然后在42摄氏度的水浴中对细胞进行热电击30秒。热休克后，向细胞中加入250微升不含抗生素的富液体培养基，并在37摄氏度下振荡孵育1.5小时。将300微升细菌悬浮液散布在一个氨苄西林Luria-Bertani平板上并孵育过夜。第二天，筛选菌落是否存在两种表型：透明和不透明。一次挑选一个透明的菌落，接种液体培养物，并在 37 摄氏度下摇匀过夜。将 150 纳克质粒、100 纳克转座酶 mRNA 和 2% 酚红在冰上混合。然后加入不含RNA的水，使体积为三微升。使用移液器将一个细胞阶段胚胎转移到充满EM的注射板中，完全覆盖琼脂，然后轻轻按压每个注射板插槽约50至75个胚胎。将mRNA质粒酚红混合物添加到毛细管注射针的开口端。将毛细管针放入注射机电枢中，然后打开注射装置。将针头放入注射板中，用镊子折断尖端，以便注射台仅泵出少量混合物。将三纳升的mRNA质粒酚红混合物注射到胚胎的细胞体中。完成后，通过轻轻地将它们从插槽中弹出，将它们从注射板中取出，然后将它们转移到带有新鲜EM的100毫米培养皿中。在28.5摄氏度下孵育。选择合适的荧光转基因表达发育阶段，并在荧光解剖显微镜下进行筛选。通过筛选荧光转基因标志物（例如由基因 Cmlc2 的心脏特异性启动子驱动的 GFP）来筛选非荧光转基因。当转基因植入阳性的胚胎发育成成虫并达到繁殖年龄时，通过将它们繁殖为野生型斑马鱼来单独筛选它们的种系传播和转基因表达能力。显示了从LR重组转化中获得的示例细菌菌落。透明菌落在85%以上的时间内含有正确的LR重组产物，而不透明菌落从未含有正确的重组产物。LR重组产物转化对三个菌落的诊断消化表明，单个不透明菌落不含任何质粒，而两个透明菌落在9， 544碱基对处含有单个条带。此处显示了 1， 950 个碱基对的转座酶 mRNA。在75%的注射胚胎中观察到镶嵌内胚层EGFP-CAAX表达。受精后24小时观察发育中心脏中Cmlc2 EGFP的转基因标志物表达。成年斑马鱼具有sox17：EGFP-CAAX的种系传播，产生荧光胚胎，内胚层完全用EGFP-CAAX标记。在对照和乙醇处理的野生型BMP突变胚胎中测量内胚层的前-后长度和每个袋从1到5个袋的背腹长度。内胚层的总长度不受基因型或治疗的影响。然而，袋一和袋三在未处理和乙醇处理的野生型胚胎之间显示出袋长度显着增加，但在未经处理和乙醇处理的BMP突变体之间显着减少长度。袋二分别显示未处理和乙醇处理的野生型和BMP突变组之间的袋子尺寸显着增加。在尝试此过程时，精确移液和稀释Tol2系统以及在胚胎注射过程中击中细胞体至关重要。

Research

JoVE Journal

Developmental Biology

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...