Name: a 3-dimensional (3d)-printed template for high throughput zebrafish embryo arraying
Uploaded: 2018-06-01T15:00:00.000+00:00
Duration: 4 min 52 s
Description: Watch this Scientific Journal Video about A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying at JoVE.com

这项工作得到了 "1000plan 青年" 计划的支持, 同济大学的创业基金, 以及21607115和21777116的自然科学基金 (Lin)。

1. 斑马鱼胚胎排列模板的设计与制作
<ol><li>
设计排列模板的 12, 8, 96 井布局, 适合一个标准的96井板。使用图 1A中列出的用于上胚卡压室的尺寸 (请参阅补充文件)。
	<ol>
<li>使用在图 1B和1D中显示的用于陷井的尺寸。</li>
		<li>使用底部真空室的图 1C中的尺寸。</li>
		<li>将图 1B中的尺寸用于空气中/插座。</li>
	</ol>
</li>
	<li>使用 3 d 打印机 (0.1 mm 精度) 打印模板;有关用于打印的推荐树脂, 请参阅材料表。 注意: 推荐使用 0.1 mm 精度的3D 打印机来制作排列模板 (请参阅材料表)。模板表面的建议颜色为深灰色或黑色。</li>
</ol>2. 斑马鱼胚胎产卵
<ol><li>在产卵前的某一天放置两对雄性和雌性鱼。用透明的塑料分隔线将雄性和雌性分开。</li>
	<li>在早上摘下分隔条, 将雄性和雌性鱼混合在一起。</li>
	<li>用细网过滤器去除雄性和雌性鱼, 并收集鱼斑马的胚胎。用250毫升的鸡蛋水冲洗胚胎 (参见材料表)。</li>
	<li>用 Holtfreter 的溶液将采集到的胚胎移植到培养皿 (直径90毫米) 上 (参见材料表), 并使用显微镜去除未受精的胚胎。</li>
	<li>将胚胎放置在28.5 摄氏度的孵化器中。在 4 h 后受精 (hpf), 观察胚胎和清除任何死亡和不健康的胚胎。胚胎现在已经准备好下一步了。</li>
</ol>3. 排列模板的制备
<ol><li>用500毫升去离子水冲洗模板2–3时间, 并把它放入烘干烤箱 (45 °c) 为5分钟。</li>
	<li>用一块密封膜 (图 2, 步骤 1) 将底部腔带入磁带。</li>
	<li>通过模板底部的风口连接真空泵。 注: 推荐的真空泵最大真空度为0.1 兆帕。要注意使用的真空强度。如果负压过强, 则在密封膜上切开一个十字形孔, 以降低压力。</li>
</ol>4. 将斑马鱼胚胎排列成96井板
<ol><li>使用塑料转移吸管, 将大约150个胚胎放置到模板中, 如图 2,步骤2所示。</li>
	<li>将真空泵与风口连接, 以在步骤3.3 中密封膜密封的腔内产生负压。</li>
	<li>水平摇动整个模板, 直到每个井都有一个胚胎被包埋 (图 2,步骤 3)。 注: 如果 Holtfreter 的解决方案在每个井中被困之前都干涸了, 在诱捕室添加额外的 Holtfreter 溶液并重复此步骤。</li>
	<li>丢弃未被埋在水井中的额外 Holtfreter 的解决方案和胚胎 (图 2,步骤 4)。</li>
	<li>关闭并断开真空泵的连接。</li>
	<li>将标准的96井板倒置到模板上 (图 2,步骤 5), 同时同时旋转两个 (图 2,步骤 6)。</li>
	<li>点击模板底部或连接空气出口到压缩气体除尘可以将所有被困胚胎从模板转移到96井板 (图 2,步骤 6)。</li>
	<li>重复步骤4.1 到 4.8, 准备额外的多井板。</li>
	<li>取下密封膜, 用500毫升去离子水从上到下清洗模板3次, 供将来使用。 注: 不要使用任何有机溶剂, 如乙醇, 清洁模板。</li>
</ol>

<ol>
	<li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Leslie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+larvae+could+help+to+personalize+cancer+treatments.">Zebrafish larvae could help to personalize cancer treatments.</a> Science. 357 (6353), 745-745 (2017).</li><li>Lin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Transformation,+Speciation,+and+Hazard+Potential+of+Copper+Particles+in+a+Model+Septic+Tank+System+Using+Zebrafish+to+Monitor+the+Effluent.">Understanding the Transformation, Speciation, and Hazard Potential of Copper Particles in a Model Septic Tank System Using Zebrafish to Monitor the Effluent.</a> ACS Nano. 9 (2), 2038-2048 (2015).</li><li>Lin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspect+ratio+plays+a+role+in+the+hazard+potential+of+ceo2+nanoparticles+in+mouse+lung+and+zebrafish+gastrointestinal+tract.">Aspect ratio plays a role in the hazard potential of ceo2 nanoparticles in mouse lung and zebrafish gastrointestinal tract.</a> ACS Nano. 8 (5), 4450-4464 (2014).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nature Communications. 4, (2013).</li><li>Lin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening+in+zebrafish+speeds+up+hazard+ranking+of+transition+metal+oxide+nanoparticles.">High content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles.</a> ACS Nano. 5 (9), 7284-7295 (2011).</li><li>Kuipers, J., Kalicharan, R. D., Wolters, A. H. G., van Ham, T. J., Giepmans, B. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+Scanning+Transmission+electron+microscopy+(nanotomy)+of+healthy+and+injured+zebrafish+brain.">Large-scale Scanning Transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.</a> Journal of Visualized Experiments. (111), (2016).</li><li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Phenotype+Recognition+for+Zebrafish+Embryo+Based+In+vivo+High+Throughput+Toxicity+Screening+of+Engineered+Nano-Materials.">Automated Phenotype Recognition for Zebrafish Embryo Based In vivo High Throughput Toxicity Screening of Engineered Nano-Materials.</a> PLoS One. 7 (4), (2012).</li><li>Tu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Categorization+and+Scoring+of+Solid,+Part-Solid+and+Non-Solid+Pulmonary+Nodules.">Automatic Categorization and Scoring of Solid, Part-Solid and Non-Solid Pulmonary Nodules.</a> in CT Images with Convolutional Neural Network. Scientific Reports. 7, 8533 (2017).</li><li>Mandrell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Lin, S., Zhao, Y., Nel, A. E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+in+vivo+model+for+nano+EHS+studies.">Zebrafish: An in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li></ol>

作者在所描述的 3 d 打印模板上填写了一项专利。

该协议中有两个关键步骤需要密切注意, 以成功地实施 3 d 打印模板排列斑马鱼胚胎。
排列模板设计中最重要的因素是诱捕井。为了确保每个井中只有一个胚胎被困, 人们应该密切关注压井的直径和深度, 以及通孔的直径。建议的直径在一个典型的胚胎 (包括绒毛膜) 直径的1.5 到2倍之内。诱捕井的深度应在典型胚胎 (包括绒毛膜) 直径的2倍以内, 以避免在同一井中堆积胚胎。通?...

斑马鱼作为一种流行的模型生物, 广泛应用于医学和毒物学领域1,2,3,4。与体外平台相比, 斑马鱼提供了更大的生物复杂性, 即一个或两种细胞无法提供。除了作为一个整体的生物模型, 斑马鱼的巨大繁殖力, 快速和同时胚胎发育, 和高器官半透明已经给这个模型独特的优势, 用于大规模毒性或药物/复合筛查5。一双成年斑马鱼每周生产的数以百计的胚胎超过了其他所有的动物模型, 并使其适合高通量筛选。
一个典型的筛选程序使用斑马鱼涉及大量的手工工作, 如成年斑马鱼产卵, 胚胎选择, 并排列胚胎进入适当的容器, 他们通过水浸泡暴露。对胚胎的发育进行监测, 可观察到的端点, 如死亡率、孵化率和异常, 通常是手动评估的, 并作为化学品毒性的初步鉴定或指示药物或化合物。为了加快筛选程序, 前面已经探讨了自动成像和计算机辅助图像分析等方法。例如, 具有高含量成像能力的显微镜已经适应了从96/384 井板6的不同发育阶段对斑马鱼胚胎进行自动亮场或荧光成像。微流控装置与显微镜结合使用, 用于定位斑马鱼幼虫通过当前操作的大脑神经元的成像7。与传统的手工操作相比, 这些方法可以显著提高图像获取的效率。此外, 在生成大量图像的同时, 还开发了图像分析工具以加快数据处理速度, 如刘et和 Tu et . 所示。8,9。
随着成像和图像分析的吞吐量水平的提高, 显而易见的是, 筛查速率限制的步骤在于准备用于暴露的斑马鱼胚胎, 这通常意味着将它们排列到96或384井板中。为解决这一瓶颈步骤, Mandrell 开发了视觉导引机器人. 10和美国11以前替换手动处理, 但这些工具相当复杂, 并且有一个深入的学习曲线来实现这些技术。因此, 提供一种易于使用的方法成为进一步提高斑马鱼筛查吞吐量水平的一个重要因素, 是这项工作的主要目标。
在这项工作中, 我们设计和制造了一个胚胎排列模板3D 打印。这种排列模板的设计, 以诱捕斑马鱼胚胎进入水井, 符合标准的96井板。而不是选择胚胎和排列他们一个一个一个一个, 你可以执行胚胎诱捕和阵列所有96胚胎成一个多层板一次。使用这个模板和下面的协议, 你可以显著提高排列胚胎的效率到多层板, 这将在短期内提高筛选能力至少比手工操作。下面描述的协议包括排列模板、斑马鱼产卵、胚胎采集和排列的总体设计。图 1显示了排列模板的总体设计。图 2显示了使用部件3和4中描述的模板的分步协议概述。

<table><tbody><tr><td>Zebrafish Facility</td><td>Shanghai Haisheng Biotech Co., Ltd.</td><td>Z-A-S5</td><td></td></tr><tr><td>Mating box</td><td>Shanghai Haisheng Biotech Co., Ltd.</td><td></td><td></td></tr><tr><td>Wash Bottle, 500 ml</td><td>Sangon Biotech</td><td>F505001-0001</td><td></td></tr><tr><td>Sodium chloride</td><td>Vetec</td><td>V900058-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10016318</td><td></td></tr><tr><td>Calcium chloride</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>20011160</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Vetec</td><td>v900182-500G</td><td></td></tr><tr><td>Methylene Blue Hydrate</td><td>TCI</td><td>M0501</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10011008</td><td></td></tr><tr><td>Sea Salts</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Pipetter</td><td>Fisherbrand</td><td>13-675M</td><td></td></tr><tr><td>Controlled Drop Pasteur Pipet</td><td>Fisherbrand</td><td>13-678-30</td><td></td></tr><tr><td>Microscope</td><td>OLYMPUS</td><td>SZ61</td><td></td></tr><tr><td>Biochemical incubator</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>LRH-250</td><td></td></tr><tr><td>3D printer</td><td>UnionTech</td><td>Lite600</td><td></td></tr><tr><td>Photosensitive resin</td><td>UnionTech</td><td>UTR9000</td><td></td></tr><tr><td>Vacuum pump</td><td>Shanghai Yukang Scientific Instrument Co., Ltd.</td><td>SHB-IIIA</td><td></td></tr><tr><td>Adhesive PCR Plate Seals</td><td>Solarbio</td><td>YA0245</td><td></td></tr><tr><td>96 well plate</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>Multi 8-channel pipette 30 - 300 &amp;mu;l</td><td>Eppendorf</td><td>3122000.051</td><td></td></tr><tr><td>Compressed Gas Duster</td><td>Shanghai Zhantu Chemical Co., Ltd.</td><td>ST1005</td><td></td></tr><tr><td>DI Water</td><td>Thermo</td><td>GenPure Pro UV/UF</td><td></td></tr><tr><td>Drying oven</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>BPG-9106A</td><td></td></tr><tr><td>System water</td><td></td><td></td><td>Water out of the facility&amp;rsquo;s water system</td></tr><tr><td>Egg water</td><td></td><td></td><td>Dilute 60mg &amp;ldquo;Instant Ocean&amp;rdquo; sea salts and 0.25 mg/L methylene blue in 1 L DI water</td></tr><tr><td>Holtfreter&amp;rsquo;s solution</td><td></td><td></td><td>Dissolve 7.0 g Sodium chloride (NaCl), 0.4 g Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;), 0.1 g Potassium Chloride (KCl), 0.235 g Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O) in 1.9 L DI water. Adjust pH to 7 using HCl and adjust volume to 2 L using Di water</td></tr></tbody></table>

a 3-dimensional (3d)-printed template for high throughput zebrafish embryo arraying

斑马鱼是全球公认的淡水有机体, 经常用于发展生物学、环境毒理学和人类疾病相关的研究领域。由于其独特的特点, 包括大的繁殖力, 胚胎半透明, 快速和同时发展,等, 斑马鱼胚胎经常被用于对化学品进行大规模毒性评估和药物/复合筛查。一个典型的筛选程序涉及成年斑马鱼产卵, 胚胎选择, 并排列胚胎成多井板块。从那里, 胚胎受到接触和化学的毒性, 或者药物/化合物的有效性可以根据表型观察相对快速地评估。在这些过程中, 胚胎排列是限制吞吐量水平的最耗时和最耗费劳力的步骤之一。在本协议中, 我们提出了一种创新的方法, 使用一个 3 d 打印排列模板加上真空操作, 以加快这一艰巨的步骤。此处的协议描述了排列模板的总体设计, 详细的实验设置和分步过程, 随后有代表性的结果。在实施时, 这种方法应证明是有益的, 以斑马鱼胚胎作为测试对象的各种研究应用。

图 3显示了典型的 3 d 打印的排列模板。本模板采用光敏树脂为原料, 由3D 打印机制成;用一层黑色颜料, 可以更好地对比胚胎的颜色。96口井 (12 8) 的位置设计为符合标准96井板。同样, 384 (24 乘 16) 井模板也可以使用相同的方法设计和制作。向上的房间稍大于标准的96井板, 以提供一个更好的配件。凹槽也被设计用来在排列期间容纳额外的胚胎。
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Watch this Scientific Journal Video about A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying at JoVE.com

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

在这里, 我们提出了一个设计和制造斑马鱼胚胎排列模板的协议, 然后详细的程序, 使用这种模板, 高吞吐量斑马鱼胚胎排列成96井板。

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

a-3-dimensional-3d-printed-template-for-high-throughput-zebrafish

Research

JoVE Journal

Environment

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

6.8K 次观看.  Tongji University. 在这里, 我们提出了一个设计和制造斑马鱼胚胎排列模板的协议, 然后详细的程序, 使用这种模板, 高吞吐量斑马鱼胚胎排列成96井板。

视频: A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

科研

发育生物学

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

斑马鱼手术尺寸缩小术中胚胎形态缩放的研究

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

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

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

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 ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

生物学

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

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斑马鱼幼虫显微注射和成像的微装置

斑马鱼手术尺寸缩小术中胚胎形态缩放的研究

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

一种简单有效的斑马鱼胚胎移植装置

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

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

多层安装于斑马鱼的长期光表镜

组织特异性荧光的斑马鱼胚胎半自动成像