Name: dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation
Uploaded: 2014-03-04T17:00:00
Description: Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

作者感谢艾德琳多莉的外植体对纤维连接蛋白，埃里克Theveneau图片的有益讨论和居里研究所的动物设施。 CM是地区法兰西岛（DOMAINE D&#39;INTERET修形茎杆），巴黎大学南基（武官Temporaire德ENSEIGNEMENT等德RECHERCHE）和法新社国立德拉RECHERCHE的博士后研究员。这项工作是由巴黎大学南基（Attractivite 2011），该中心法国国家科学研究（动作Thematique等Incitative河畔计划），协会倒拉RECHERCHE CONTRE勒癌症格兰特SFI20101201882，法甲CONTRE乐癌症和法新社国立德拉RECHERCHE（法新社国立德拉RECHERCHE计划布兰克）。

实验符合的用于科学目的和更换，减少和细化的国际公认原则的动物的保护国家和欧洲的监管。 1。纤连蛋白包被的培养皿14的制备<ol><li>移液器500毫升的10毫克/毫升培养级纤连蛋白稀释在1×磷酸盐缓冲盐水（PBS）中插入标准塑料培养皿中（直径40×11毫米）。在37℃下1小时。注意：如果使用玻璃餐具或盖玻片后续成像，使用100毫克/毫升纤维连接蛋白的解决方案。 </li>...

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本协议描述了一个简单的方法来植体premigratory颅NC在非洲爪蟾胚胎。用于这些实验的胚胎必须健全和愈合良好。丢弃任何批次不健康的胚胎。此外，胚胎生长在不同的温度（12〜18〜20℃），以切除神经嵴在阶段17，而不是以后。阶段17后，神经嵴可与头侧中胚层混合，并不能完全除去。 爪组织迅速愈合，然后失去其粘附到其他组织。它能够快速工作，并把神经嵴植入宿主，只要它是从...

神经嵴（NC）是一个短暂的胚胎细胞群出现从神经管在神经胚形成在脊椎动物胚胎的末端。控制NC规范的信令和遗传事件早原肠胚形成开始。 NC被指定在神经和非神经外胚层通过从周围背部组织的信号之间的边界。在神经胚形成的端部，NC细胞经历了上皮至间质转化（EMT），并广泛地在胚胎移植以下刻板航线通过响应周围指导线索。一旦他们达到他们的最终目的地，它们分化 ​​成衍生物， 如神经元，神经胶质细胞，骨，软骨和色素细胞1-5繁多。因为他们有许多类型的细胞和胚胎组织，在数控细胞发展的任何步骤中的缺陷，从感应到最终分化的贡献，可引起命名neurocristopathies 6先天性综合征。 Ë发展数控xperimental操作在不同的阶段 - 规范，EMT，迁移和分化 - 将提高我们neurocristopathies的认识，并允许潜在的治疗策略设计。 非洲爪蟾胚胎选择的模型来研究数控的发展。大量的胚胎很容易获得，并且体外受精可进入发展的第一个步骤。许多工具可用来操纵实验十蟾胚胎发育。基因增益的功能和击倒很容易通过显微注射的早期囊胚单个细胞来执行。胚胎组织可切体外复性7-11或背嫁接试验12,13。 在这个协议中，我们描述了如何解剖出premigratory颅NC在十蟾晚neurulas，迁移之前。这些外植体可以在纤连蛋白金属线进行培养D平板学习迁移和分化的控制， 在体外实验条件。数控植，也可嫁接到正常或操纵宿主胚胎，以研究它们的迁移和分化的体内 。

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			<td height="20" style="height:20px;width:244px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F4759-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bovine Serum Albumine. Fraction V</td>
			<td style="width:71px;">Euromedex</td>
			<td style="width:119px;">04-100-811-C</td>
			<td>Lower quality grade may&#160; be suitable for this application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
			<td style="width:71px;">FST</td>
			<td align="right" style="width:119px;">26001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
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</table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

神经嵴（NC）是一个短暂的背神经管细胞群，在神经胚形成年底经历了一个上皮至间质转化（EMT），广泛迁移对各个器官，并分化成多种类型的衍生工具（神经元，神经胶质细胞，软骨和骨，色素和内分泌细胞）。在这个协议中，我们描述了如何从非洲爪蟾胚胎解剖premigratory颅数控，以研究开发数控在体内和体外 。青蛙模型提供了许多优势，研究开发初期，大量批的情况下，胚胎发展迅速， 在体内增益和功能丧失的策略允许操纵基因表达前，数控夹层供体和/或主机的胚胎。数控植体可以镀在纤连蛋白和用于体外研究。它们可以在无血清的定义培养基中培养数天。我们还描述了如何嫁接数控植回到宿主胚胎研究的NC迁移和分化的体内 。

当镀上纤连蛋白，神经嵴外植体附着快速（15-30分钟）和植传播平面内2小时（ 图1A）。经过3-6小时的细胞开始分散。在24小时后在15℃下，许多细胞已开始迁移远离外植体（ 图1B和1C）。蛋黄使得细胞在相衬（ 图1B）非常明亮。细胞突起（filopodes和lamellipodes）都清晰可见鬼笔环肽与肌动蛋白细胞骨架（ 图1C）染色后。 <p class="jove_cont...

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Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

本协议描述了如何从非洲爪蟾 neurulas解剖premigratory颅神经嵴（NC）。这些外植体可以镀在纤连蛋白和体外培养，或接枝回主机胚胎。这种技术可以让学习数控上皮至间质转变，迁移和分化的机制。

剖析<em&gt;非洲爪蟾</em&gt;神经嵴的<em&gt;在体外</em&gt;外植体培养或<em&gt;在体内</em&gt;移植

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Research

JoVE Journal

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

Plastic Embedding and Sectioning of Xenopus laevis Embryos

塑料嵌入和非洲爪蟾胚胎切片

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

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 ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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

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

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...