Name: tissue determination using the animal cap transplant (act) assay in xenopus laevis
Uploaded: 2010-05-16T17:00:00
Description: Watch this Scientific Journal Video about Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis at JoVE.com

这项工作得到了从研究经费，以防止失明（职业发展奖MEZ和ASV和不受限制授予眼科部），大肠杆菌明德Zeigler基金会（MEZ和ASV）和美国国家眼科研究所/美国国立卫生研究院（MEZ ，补助金R01EY015748和R01EY017964）。

第一部分设置为ACT检测<ol><li>生成皑皑的RNA注射制造商的建议，使用他们的酚：氯仿纯化方法（如mMessage mMachine套件;美国应用生物系统公司/ Ambion公司，奥斯汀，德克萨斯）。 RNA的编码一种荧光蛋白作为示踪。我们抄录或者黄色荧光蛋白（YFP）或mCherry 5基因，这是在pCS2 +表达载体。 </li><li>拉硼硅玻璃毛细管形成锥形玻璃提示，使用微量拉马。我们使用萨特微管牵引器，P - 97（萨特仪器公...

<ol>
	<li>Smith, W. C., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin,+a+new+dorsalizing+factor+localized+to+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Lamb, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+by+the+secreted+polypeptide+noggin.">Neural induction by the secreted polypeptide noggin.</a> Science. 262, 713-718 (1993).</li><li>Green, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Methods+in+Developmental+Biology:+Xenopus+&#38;+Zebrafish.">Molecular Methods in Developmental Biology: Xenopus &#38; Zebrafish.</a> Methods in Developmental Biology. , (1999).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7, e1000174-e1000174 (2009).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J Vis Exp. , (2008).</li><li>Lan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+Elicits+Retinal+Fate+in+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate in Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. , (2009).</li></ol>

在这段视频中，我们已经证明我们的爪生物学家使用的经典技术，我们重新安排，以建立动物帽移植的实验版本。在室温条件下， 爪蟾胚胎发育非常迅速地通过9级和15级。通过放置在14℃，胚胎发育减慢，并可以在一个实验中进行更多的移植。如果有必要将增长到老年阶段（42/43）的胚胎，那么我们建议，利用转基因金星YFP的动物，因为年纪较大的蝌蚪注入荧光蛋白在信号衰。我们?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>mMessage mMachine SP6 Kit</td>
<td>&nbsp;</td>
<td>Applied Biosystems/Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate Capillary Tubes (1.0mm OD x 0.5mm ID)</td>
<td>&nbsp;</td>
<td>FHC, Inc</td>
<td>27-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Sutter Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments, Inc.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin sulfate [50 mg/ml]</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BW17-528Z</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard/elastomer Kit 1.1lb</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9644388</td>
<td>&nbsp;</td>
<tr>
<td>60 x 15 mm polystyrene petri dish</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>&nbsp;</td>
<tr>
<td>human chorionic gonadotropin</td>
<td>&nbsp;</td>
<td>Intervet</td>
<td>22219</td>
<td>&nbsp;</td>
<tr>
<td>Pico-Injector Microinjection Systems</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>650003</td>
<td>&nbsp;</td>
<tr>
<td>Torrey Pines Scientific ECHOtherm Incubator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-680-21</td>
<td>&nbsp;</td>
<tr>
<td>Transfer pipette</td>
<td>&nbsp;</td>
<td>Krackeler</td>
<td>6290-20635A</td>
<td>&nbsp;</td>
<tr>
<td>Dumostar #5 Forceps</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11295-10</td>
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tissue determination using the animal cap transplant (act) assay in xenopus laevis

许多蛋白质​​在胚胎发育中发挥的双重作用。那些规范在一个特定的组织细胞命运决定的，也可以影响的一个较大区域的胚胎的发展。这使得确定其在一个特定的组织中的作用难以分析。例如，<em&gt; noggin</em&gt;过度表达<em&gt;非洲爪蟾</em&gt;胚胎，导致整个前地区的扩张，包括眼<sup&gt; 1,2</sup&gt;。从这样的结果，它是不知道，如果Noggin次在眼睛的决心，或直接作用，导致神经组织的扩张，Noggin间接影响黑眼圈的形成。在这种复杂的表型，研究它的眼睛特定的细胞命运决定的作用难以分析。我们已经开发了一个实验，克服了这一问题。趁着多能干的性质<em&gt;非洲爪蟾</em&gt;动物帽<sup&gt; 3</sup&gt;，我们开发了一种检测基因产物的能力测试（S），如<em&gt; noggin</em&gt;或眼域的转录因子（EFTFs），转换成特定的组织或细胞类型一侧的胚胎移植到这个组织的上限<sup&gt; 4</sup&gt;。虽然我们发现无论是Noggin蛋白治疗或转录因子的集合，可确定在动物帽视网膜细胞的命运，这个过程可以用来识别指定其他组织以及所涉及的基因产物（S）。

Watch this Scientific Journal Video about Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis at JoVE.com

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

动物帽高表达的基因产物（S）移植到发展中国家的侧翼<em&gt;非洲爪蟾</em&gt;胚胎，以建立组织是否确定。

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 ...

Research

JoVE Journal

Biology

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

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 ...

Mammary Epithelial Transplant Procedure

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

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 ...

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. ...

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

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

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 ...