Name: methods for tattooing xenopus laevis with a rotary tattoo machine
Uploaded: 2024-06-28T15:00:00.000+00:00
Duration: 4 min 18 s
Description: Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling ...

我们感谢达特茅斯学院的比较医学和研究中心为本协议中使用的动物提供日常饲养。我们还感谢 Leah Jacob 和 Adwaita Bose 在测试协议和拍摄动物方面的帮助。最后，我们感谢 Ann Miller 的实验室在纹身实践方面的培训。这项工作得到了 NIH 赠款 R00 GM147826 JL 的支持。

所描述的所有动物程序均已获得达特茅斯学院机构动物护理和使用委员会的批准。
1. 设备设置
注意:包括该程序的工作流程和示例工作台设置（图 1）。
<ol><li>将纹身枪和脚踏板连接到电源。将脚踏板放在工作表面下方。</li>
	<li>组装纹身枪<ol><li>使用内六角扳手或六角螺丝刀，松开手柄中的螺丝（图 2A，以红色圈出）。</li>
		<li>将塑料尖端完全插入手柄中。拧紧螺钉以将其固定到位。</li>
		<li>将金属管插入手柄的背面（图 2A）。这将进行调整，因此请将其插入到其长度的一半。拧紧螺钉以将其固定到位。</li>
		<li>将纹身针穿过手柄的背面，使其舒适地位于手柄前部的塑料尖端中（图 2B）。</li>
		<li>将针头和金属管的背面滑过管夹。调整金属管，使针头舒适地安装在塑料尖端中（图 2B）。用手拧紧管夹，直到管子无法移动（图 2C）。</li>
		<li>取下黑色 O 形圈，将针钩在转子臂上;更换 O 形圈以将针固定到位（图 2C）。</li>
	</ol></li>
	<li>设置电源上的电压，使有足够的功率刺穿皮肤，但不要太强大以至于难以控制。插入并连接电源后，踩下脚踏板以确保机器正常工作。注意不要将针头指向您自己或其他人员。 注意:本协议中使用的纹身机在 6.0 和 9.0 V 之间工作最佳，但这可能因纹身机和电压电源而异，应根据经验确定。</li>
	<li>用黑色纹身墨水填充 1.5 mL 微量离心管或提供的塑料墨水罐的盖子，直至装满约四分之三。</li>
</ol>2. 麻醉
<ol><li>在一个足够大的水箱中准备麻醉剂，以浸入一只成年雌性 X. laevis 青蛙。<ol><li>使用对青蛙安全（无氯）的水，加入三卡因至终浓度为 1.5 g/L，加入碳酸氢钠至终浓度为 3.5 g/L。该溶液的 pH 值为 7.15。 注意:三卡因是一种刺激物。</li>
	</ol></li>
	<li>将一只青蛙浸入麻醉罐中。确保青蛙浸没在三卡因溶液中，直到麻醉（7-8 分钟）。<ol><li>要检查青蛙是否完全麻醉，请将其捡起来，倒置，然后用一只脚用力挤压它。如果青蛙没有退缩或反应，就可以纹身。</li>
		<li>如果青蛙始终需要 10 分钟以上的麻醉时间，请准备用碳酸氢钠缓冲的新鲜三卡因溶液。 注意:青蛙在三卡因溶液15 中的停留时间不应超过 30 分钟。</li>
	</ol></li>
</ol>3. 纹身
注意:在给活体动物纹身之前，在一块果皮坚硬的水果（如柠檬或香蕉）上练习会很有用。
<ol><li>纹身前为青蛙准备一个回收罐。在水箱中装满 10-15 升新鲜的青蛙安全水，并添加一个聚苯乙烯泡沫塑料岛。这将为青蛙提供一个表面，让青蛙醒来而不会溺水。 注意:可以使用放置在拉链袋内的聚苯乙烯泡沫塑料冷藏集装箱的盖子制作聚苯乙烯岛。</li>
	<li>将麻醉的青蛙仰卧在干纸巾或工作台纸上（图 3A）。使用未漂白的（棕色）纸巾进行所有涉及青蛙的工作。</li>
	<li>使用干燥的无绒湿巾擦去青蛙胸部的水和粘液。</li>
	<li>用手指找到青蛙胸部中央的胸骨，然后用非惯用手拉紧皮肤。</li>
	<li>标记青蛙<ol><li>相对于工作表面垂直握住组装好的纹身枪，然后将针头浸入墨水中（图 3B）。</li>
		<li>保持组装好的纹身枪垂直，在踩下脚踏板之前，将针尖压在青蛙的皮肤上。在青蛙的皮肤上画线，同时施加均匀的压力。 注意:一些轻微的出血或发红是正常的，轻微的皮肤脱落也是正常的。</li>
		<li>如果针头卡在青蛙的皮肤上，请用湿巾清除针头上多余的墨水或皮肤。纹身时墨水过多是正常的。</li>
		<li>如果青蛙的胸部被太多墨水覆盖，无法清楚地看到被标记的区域（图 3C），请用青蛙安全的水和湿巾清除多余的墨水（图 3C'）。然后，用干布吸走水分并继续上墨。 注意:纹身前可以准备一瓶对青蛙安全的水。</li>
		<li>继续在同一区域上墨，直到擦去多余的墨水后，仍然留下深色、清晰的数字（图 3D）。不要在一个笔触中绘制整个标记或数字，而是重复创建较小的笔触，尤其是对于曲线。</li>
	</ol></li>
</ol>4. 恢复
<ol><li>用蛙类水润湿纸巾，然后将其平放在聚苯乙烯泡沫塑料岛的顶部（图 4A）。</li>
	<li>将青蛙放回站立的水箱中<ol><li>将青蛙腹部朝下放在面向水的毛巾上（图 4B）。将纸巾的一半折叠在青蛙的后半部分，然后用杯状手弄湿青蛙的顶部。一旦青蛙从麻醉中苏醒（纹身后约 1 小时），它们将独立入水中并正常游泳。</li>
		<li>纹身完全愈合后，24 小时后将青蛙送回长期住房。</li>
	</ol></li>
</ol>5. 清洁和设备维护
<ol><li>重复使用和高压灭菌纹身针<ol><li>用 100% 异丙醇和去离子水冲洗针头，使墨水松动，擦去剩余未溶解的墨水。</li>
		<li>擦洗后，将针头放入金属高压灭菌箱中，或用铝箔包裹并高压灭菌 30 分钟，干燥循环。</li>
	</ol></li>
	<li>如果纹身机或其他设备上的碎片上有墨水，请用 100% 乙醇和纸巾擦拭。</li>
	<li>麻醉溶液的最终三卡因浓度约为 0.5%。根据机构危险废物管理指南对其进行处理。</li>
	<li>设备存放和维护<ol><li>如果可能，请将纹身机存放在干燥的房间里。过多的水分会损坏电子元件并缩短机器的使用寿命。</li>
		<li>为了保持纹身机处于良好的工作状态，请检查转子上的轴承（图 2C）以确保它没有因振动22 而松动。这可以用内六角扳手或六角螺丝刀拧紧。</li>
		<li>大约每月一次，使用浓稠的润滑脂22 润滑转子臂上的轴承。</li>
		<li>在一些纹身机上，转子臂可能会卡住并且不会振动。要纠正此问题，请扭转转子臂或压缩弹簧，使针处于降低位置（图 2C）。</li>
	</ol></li>
</ol>

优化两栖动物标记:非洲爪蟾纹身指南

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作者声明没有利益冲突。

纹人是一种可以追溯到几千年前的艺术形式，只要人类给自己纹身，他们就给动物纹纹身或打上烙印23。用于标记动物（尤其是哺乳动物）的设备和技术已经成熟、有据可查且广泛可用。虽然标记动物最初是为了区分牲畜和防盗23，但它在生物医学研究中的作用也变得同样重要。
两栖动物纹身的一个好处是纹身会在 24 小时内愈合，好的纹身...

动物模型是研究与人类健康有关的问题的有用工具。在实践中，使用动物模型的生物医学研究需要仔细组织和维护健康的动物群体。合乎道德的动物处理和饲养的最佳实践旨在减少实验所需的动物数量并改进实践以确保动物福利1.爪蛙属，包括 Xenopus laevis （X. laevis;非洲爪蛙）和非洲爪蟾 （X. tropicalis;西部爪蛙）自 1930 年代以来一直被用于生物医学研究，当时南非医生使用 X. laevis 进行第一次妊娠试验2。虽然现代妊娠试验不再需要青蛙，但非洲爪蟾研究的作用仍然存在。使用非洲爪蟾进行生物医学研究的优势包括注释良好的基因组3 （genomes）、大窝卵的全年诱导排卵4 以及适合体外受精的外部产卵。这些特征使它们成为脊椎动物胚胎学和发育 5,6,7、基本分子和细胞生物学 7、8、9、10 以及人类疾病建模 7,11,12,13 的有用资产。
追踪个体 非洲爪蟾 动物的可靠方法对于记录生物重复和提高研究的严谨性和可重复性至关重要。由于 非洲爪蟾 经常成群结队地饲养，动物标记使研究人员能够轻松追踪个体动物4。保持动物的准确记录可以节省时间和资源，并提高跟踪动物健康状况的能力。例如，动物的个体鉴定可以改进生成转基因 非洲爪蟾 品系的组织工作流程，因为这需要通过测序14 验证具有特定基因型的多代青蛙，这需要对动物进行组织和个体鉴定。当这些突变缺乏易于辨别的成体表型时，尤其如此。同样，使用 非洲爪蟾 卵母细胞和胚胎来研究基本的细胞和发育生物学也受益于跟踪个体动物。诱导排卵后，动物需要休息至少 3 个月，以防止出现高排卵综合症等健康并发症15。个体鉴定方法可确保动物不会被诱导过于频繁地排卵。
标记和跟踪动物还使实验室工作人员能够跟踪动物的健康问题。相同基因型的动物生病可能表明过度近亲繁殖或与转基因相关的意外健康问题。同样，动物在近期排卵后生病可能表明试剂、材料或技术存在问题。跟踪动物及其健康状况使实验室工作人员和兽医能够在问题再次出现时进行跟进，并采取预防措施来预防未来的疾病。在哺乳动物中，有许多鉴定方法。小鼠的永久性方法包括打耳、耳标、纹身和皮下微芯片16。这些可以清晰可靠地区分菌落或笼子内的动物，并且可以由实验室人员轻松管理。打耳等方法是微创的，只需要一件专用设备，适用于大多数年龄段的动物。虽然这些系统对小鼠来说简单且有用，但它们在青蛙中的使用带来了一系列独特的挑战。青蛙和其他两栖动物没有耳廓（外耳结构）。一些研究人员在动物的下巴、脚趾或后肢上贴上了标签17,18。这种方法导致了各种问题:颌标签会引起刺激，激动的青蛙试图用前肢拉下标签17。脚趾标签刺穿了脚趾之间的蹼，影响了运动并有迷路的风险。因此，两栖动物需要自己的识别方法。从历史上看，剪脚也被用来标记两栖动物17,19。脚趾用一把锋利的剪刀剪住，可以通过前脚和后脚的脚趾长度或脚趾向后长出的角度来识别动物（在蝾螈中）。然而，这种方法提出了道德问题，即剪脚趾可能会损害动物的运动17。此外，这会导致出血并带来感染风险。另一个已建立的标记系统是皮肤自体移植物，其中皮肤从青蛙的一部分取出并通过手术连接到另一部分。例如，描述了一种使用来自胸部的浅色皮肤移植物标记青蛙背部或肩部的方法20.皮肤移植也有局限性和风险:该手术是侵入性的，并引入了嗜水气单胞菌感染或红腿的风险，这是一种可能致命的疾病;自体移植物的完全愈合需要长达 6 周;并且使用所描述的方法，由于放置自体嫁接20 的位置有限，只能将 6 只青蛙饲养在一起。
微创标记方法包括玻璃珠和应答器芯片17,19。在玻璃珠法中，玻璃珠被拧到一个小缝合线上，然后缝在青蛙的皮肤上。这比皮肤自体移植物提供了更大的可变性，至少有 60 种不同的颜色组合。然而，缝合线可能会脱落并导致珠子丢失。或者，可以将微芯片应答器植入青蛙背侧淋巴囊的皮肤下。这被认为是最持久的标记方法，可以单独识别和编目无限数量的动物。然而，这也是最昂贵的方法，因为单个微芯片很昂贵，而且标记大菌落的成本很高。微芯片还需要一个特殊的扫描仪来读取19。非洲爪蟾鉴定的一种常见方法是参考动物的自然颜色和图案。这对 X. laevis 等青蛙特别有效，它们在整个成年期都有明显的模式。然而，这些模式会随着时间的推移而随着压力而变化，当青蛙在透明和有色容器之间移动时，颜色会显得不同15。此外，这种鉴定方法对 X. tropicalis 的用处较小，与 X. laevis 相比，X. tropicalis 的标记模式不那么明显，或者对于没有颜色标记的白化动物21。即使对于具有不同标记的物种，实验室工作人员也可以以不同的方式解释标记的位置和大小，这可能会导致识别错误。因此，结合额外的识别方法对动物进行拍照是最可靠的。因此，我们寻求使用一种易于识别、永久且侵入性最小的技术来标记和识别非洲爪蟾动物。
描述两栖动物纹身方法的已发表资源有限。纹身已与其他品牌技术一起被描述，包括热品牌、硝酸银品牌和冷冻品牌17。在同一资源中，纹身是通过用 27G 皮下注射针头绘制数字来完成的，并且注意到该过程不会引起感染，这与其他品牌技术形成对比，后者使用形状为数字或其他标记的金属丝。在另一个来源中，使用电动纹身机（描述为振动针）来标记青蛙，但关于该技术的细节很少17,19。作者警告说，通过扰乱青蛙的保护性粘液层，这个过程会增加红腿的风险。虽然没有一种标记或识别方法既是完全无创的（如照片）又是永久性的（如微芯片），但纹身提供了一种有效的折衷方案。与其他技术（例如皮肤自体移植）相比，纹身相对简单。其他好处包括较小的学习曲线和相对便宜的设备。给水生两栖动物纹身会带来一定的挑战，这可能会吓到研究人员并损害成功的动物标记。本文旨在为研究人员提供有据可查的用旋转纹身机为成年非洲爪蟾纹身的方法。

<table><tbody><tr><td>3 needle round liners</td><td>Worldwide Tattoo Supply</td><td>1203RLB</td><td>Packaged sterile</td></tr><tr><td>5 Needle Round Disposable ULTRA</td><td>Worldwide Tattoo Supply</td><td>HTIPRS5-U</td><td>Packaged sterile</td></tr><tr><td>5 needle round liners</td><td>Worldwide Tattoo Supply</td><td>1205RLB</td><td>Packaged sterile</td></tr><tr><td>7 needle round liners</td><td>Worldwide Tattoo Supply</td><td>1207RLB</td><td>Packaged sterile</td></tr><tr><td>Clip Cord</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Foot pedal</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Inkpots</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Kimwipes, delicate task wipes</td><td>Fisher Scientific</td><td>06-666A</td><td></td></tr><tr><td>RCA Connection</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Scream Ink Pitch Black, 1oz</td><td>Worldwide Tattoo Supply</td><td>SI101</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO3)</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Stainless steel grips</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Stealth 2.0 Rotary Tattoo Machine</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Stealth 2.0 Rotary Tattoo Machine Box Set</td><td>Worldwide Tattoo Supply</td><td>STEALTH2-SET</td><td></td></tr><tr><td>Styrofoam island</td><td>N/A</td><td>N/A</td><td>This is the lid of a styrofoam cold shipping container</td></tr><tr><td>Tricaine (ethyl 3-aminobenzonate methanesulfate)</td><td>Sigma-Aldrich</td><td>E10521</td><td>CAUTION: IRRITANT</td></tr><tr><td>Unbleached paper towels</td><td>Grainger</td><td>2U229</td><td>Paper towels MUST be unbleached, bleach is toxic to amphibians</td></tr><tr><td>Voltage Supply</td><td>Worldwide Tattoo Supply</td><td>N/A</td><td></td></tr><tr><td>Wash bottle (with frog-safe water)</td><td>Fisher Scientific</td><td>FB0340923T</td><td>Frog safe water is dechlorinated, pH 7.0-8.5, conductivity 1200-1800 uS</td></tr><tr><td>&lt;em&gt;X. laevis&lt;/em&gt; adult female</td><td>Xenopus1</td><td>N/A</td><td></td></tr><tr><td>Zip-top plastic bag</td><td>N/A</td><td>N/A</td><td>This bag should be large enough to hold the styrofoam island</td></tr></tbody></table>

methods for tattooing xenopus laevis with a rotary tattoo machine

动物模型扩大了生物医学研究的范围，进一步加深了我们对发育、分子和细胞生物学的理解，并使研究人员能够对人类疾病进行建模。记录和跟踪个体动物使研究人员能够减少研究所需的动物数量，并改进实践以改善动物福祉。有几种有据可查的标记和追踪哺乳动物的方法，包括打耳和耳标。然而，标记水生两栖动物物种的方法有限，现有资源过时、无效或成本高得令人望而却步。在这份手稿中，我们概述了用旋转纹身机标记 非洲爪蟾 的方法和最佳实践。适当的纹身可以产生高质量的纹身，使研究人员很容易区分个体，并且对动物健康构成的风险最小。我们还强调了纹身质量差的原因，这会导致纹身迅速褪色并对动物造成不必要的伤害。这种方法允许研究人员和兽医对两栖动物进行标记，使他们能够跟踪生物重复和转基因品系，并准确记录动物健康状况。

高质量的纹身会在青蛙的胸部有深色、清晰的笔触，并且可以在几英尺外清楚地区分出来（图 5A）。一般来说，较大的数字和标记更便于阅读，但较长的名称和数字可以做得更小，以便舒适地贴合青蛙的胸部。纹身的寿命更难判断，但高质量的青蛙纹身应该至少保持 3-6 个月的深色和清晰可辨（图 5D-D'）。一旦纹身褪色，就可以使用相同的...

作者聚焦:改进用于生物医学研究的 非洲爪蟾 标记技术

在这里，我们描述了用旋转纹身机为成年非洲 爪 蟾（非洲爪蛙）纹身的方法。适当的纹身会产生深色、易于辨认的数字，这些数字可以持续数月，并使动物易于区分，以便进行研究和记录。

用旋转纹身机纹非洲 爪蟾 的方法

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling ...

methods-for-tattooing-xenopus-laevis-with-a-rotary-tattoo-machine

Research

JoVE Journal

Biology

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

591 次观看.  Dartmouth College. 我们的研究重点是使用非洲爪蟾卵提取物和支持脂质双层来研究细胞皮层的组织。我们正在努力了解动态细胞骨架模式如何在皮层内受到调节，以及这些模式如何支持细胞分裂等基本细胞功能。该协议概述了一种有效且安全的方法来标记非洲爪蟾。这种方法使研究人员能够追踪遗传系和实验重复，并监测动物健康状况。纹身是一种具有成本效益...

视频: 作者聚焦:改进用于生物医学研究的 非洲爪蟾 标记技术

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

生物工程

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

生物学

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

神经科学

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

用旋转纹身机纹非洲爪蟾的方法

副本

摘要

探索更多视频

此视频中的章节

利用共聚焦分析

技术目标显微注射到显影

皮肤纹身作为一种新的方法DNA疫苗交货

胚胎血管生成的可视化和定量分析

非洲爪蟾卵母细胞的微量注射

的神经植体培养

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

用旋转纹身机纹非洲 爪蟾 的方法

副本

摘要

探索更多视频

此视频中的章节

利用共聚焦分析

技术目标显微注射到显影

皮肤纹身作为一种新的方法DNA疫苗交货

胚胎血管生成的可视化和定量分析

非洲爪蟾卵母细胞的微量注射

的神经植体培养

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

用旋转纹身机纹非洲爪蟾的方法