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（萨特仪器公司，诺瓦托，CA），如前面所述6。 </li><li>表1无菌解决方案，从一个10 X马克的修改林格的疫苗（MMR）解决方案的股票（10 mM的MgCl 2的20 mM的氯化钾; 20 mM 的氯化钙; 50 mM的HEPES; 1中号氯化钠;调整pH值7.5，高压灭菌，并储存在RT）。 </li><li>隔离从一个男性的青蛙睾丸和组织1X MMR /庆大霉素（50微克/毫升硫酸庆大霉素; BioWhittaker，猫＃17 - 518Z）在无菌溶液保存在4 ° C。这可以在实验开始前的一个星期，但它是失去时间的推移其疗效。 </li><li>产蛋的解决方案（1X：2.62 mM的氯化钾，氯化钠150毫米，0.8毫米的Na 2 HPO 4 Trizma基地，20毫米，2.62 毫米碳酸氢钠3，2.75毫米硫酸镁;调整pH至7.6），可作为8X股票，这可以保存在4 ° C为两个星期。 注意 ：要注意在这一解决方案的污染，因为这会影响鸡蛋的质量。 </li><li>生成注入板块。要做到这一点，在0.4X在微波炉MMR中熔化1％琼脂糖，加入到60毫米板〜6毫升熔化的琼脂糖溶液，轻轻解开一个弹性模具上顶级的解决方案，以防止泡沫，让凉爽约（图1）在室温下20分钟。要确定所需的数量，考虑实验条件（ 例如：1，YFP的; 2，YFP + Noggin）和需要移植的主机胚胎数量。对于我们的实验中，我们为每个实验条件下产生至少20移植的主机胚胎。在18-19℃，胚胎将在阶段15，约一个小时。如果需要一小时进行20次移植，则需要两个施肥时间点，假设主机的胚胎接受的移植。因此，将需要四个注射菜（2 EXP条件× 2施肥时间点= 4）。长达一个星期，保存在4 ° C的板块。 </li><li>生成第隔离板使用1％琼脂糖+ 0.7X MMR疫苗注射板与模具。请注射板加一个额外的每施肥时间点（4 +2施肥时间点= 6）相同数量的上限隔离。 </li><li>注入500个单位绒毛膜促性腺激素显微注射前一天在一天中较晚的女性。商店女性在16-18 ° C过夜。 </li></ol>第二部分。 非洲爪蟾胚胎的RNA显微注射 A.制备用于显微注射<ol><li>取出女性激素诱导它们放置在个人坦克包含1X产蛋解决方案（ELS）注 ：我们不使用已在ELS两个多小时，卵子的质量，减少鸡蛋。因此， 在体外受精前约一个小时，我们放弃从油箱，使所有的鸡蛋只刚产下的蛋收集。 </li><li>准备通过剪断尖获得直径为25-35微米的显微注射针。广场上picoinjector针和校准，使分配每次注射10 NL。 </li><li>设置一个孵化至14 ° C。 </li><li>一旦女性奠定了约一个小时，使用移液管从水箱取出鸡蛋和60毫米培养皿放入鸡蛋。鸡蛋尽可能多的ELS删除和替换1X的MMR。受精鸡蛋可以马上或在1X的MMR逗留长达一个小时。对于移植实验，这是最好的施肥两个菜，80％的鸡蛋最多的单层三个分批于上午11时左右，中午和下午1时。注射后，它们将被放置在14 ° C，使他们成长到第隔离（阶段8.5-9）和器官移植所需的阶段（第一阶段15;见我，＃6段）部分。 </li><li>要检查睾丸的质量，我们执行一个清晨施肥（上午8时），它允许我们提取从另一个男性的睾丸，如果从第一个精子是没有生命力的的。注：我们均质〜0.25睾丸在1 mL 1X孕产妇死亡率和增加约5至7％的卵母细胞板滴。 </li><li>匀浆睾丸在体外授精的鸡蛋，一个小时后，使用果冻解决方案上的鸡蛋取出果冻大衣（164μl1M数码地面电视+ 10ml 1M三中，pH值8.8至50毫升蒸压nanopure水） 。等待，直到他们达到两细胞阶段开始排序，这需要约2小时18 ° C或1.5小时，在室温（22℃）。 </li><li>使用一个TRANSF二吸管，排序均匀色素和均匀分裂的胚胎，作为显示在图3中左边的动物帽，到充满0.4X孕产妇死亡率+ 6％聚蔗糖注射菜很好。 </li></ol> B.微量注射<ol><li>长达一个60毫米的培养皿盖顶部的封口膜。吸取2μL上的封口膜和使用PLI - 100来填补你的注射针吸你的第一个RNA样品。一定要检查样品针仍然是配药开始前注射液10 NL。 </li><li>在另一盘7 500 PG YFP的RNA在一盘和您感兴趣的（如20 PG Noggin RNA）的基因加YFP的RNA注入20-30两细胞阶段的胚胎（两个卵裂球）。 </li><li>广场100-200非注射胚胎，每个时间点，在另一个与聚蔗糖溶液注射菜。将它们和孵化器，同时注入的胚胎。这些都是要在第四部分中使用的主机。 </li><li>注射后，所有胚胎孵育14℃过夜。重复所有的时间点。 </li></ol>第三部分。隔离注入胚胎的动物帽<ol><li>清晨抵达阶段的胚胎。最早的时间点应该是第9期。删除那些没有生存。继续执行以下步骤，每个时间点分批工作。 </li><li>从每盘倒掉的聚蔗糖的解决方案，并添加回0.7X MMR /庆大霉素解决方案。一个上限隔离菜，加0.7X MMR /庆大霉素解决方案的慷慨金额。使用移液管和地方在新鲜配制的第隔离菜一个，从他们的注射菜井打跑胚胎。 </li><li> 0.7X孕产妇死亡率+庆大霉素溶液中分离的动物帽，保持完整的分期2个或3个胚胎。无论是Gastromaster，安装有一个黄色的提示弯曲至400微米，或一双锐利5钳都可以使用。 </li><li>直到第二天将在14 °彗星孵化器盖。孵化器在为上限的同时，保持在相同的供体组织的发育阶段，他们应该从主机胚胎。 </li></ol>第四部分。移植到侧翼上限<ol><li>从第一个时间点从14℃培养箱取出瓶盖和胚胎阶段的胚胎。统一的黄色或红色荧光阳性的上限，根据示踪剂注入（图2），排序。 </li><li>计数的上限数量，删除双阶段12-13胚胎与0.7X MMR /庆大霉素溶液新鲜配制的注射菜。随着尖锐的＃5钳，删除每个卵黄膜。他们转移到该板块的上限，但要小心。如果没有他们的卵黄膜，胚胎可以快速解离水面。 </li><li>使用gastromaster（黄尖，200 - 250微米的弯曲线，最高设置）或＃5镊子，取出一个200微米见方一侧的胚胎阶段14日至15日（图3）。然后，削减了一半的上限（与gastromaster或＃5镊子）和地方组织在主机中的胚胎中取得的孔。旋转以及对胚胎允许愈合。许多胚胎，你可以做，而他们在舞台15。重复所有的时间点。 </li><li>胚胎通常会在30分钟内愈合。删除的死细胞（白）＃5镊子轻柔的刷牙议案。养在18 ° C过夜。 </li><li>荧光显微镜下解剖，荧光标记，移植组织的排序胚胎。把它们早在18 ° C至成长，直到42-43阶段。 </li><li>在tricaine麻醉动物。在这一点上，你可以采取的荧光解剖显微镜下的图片。完成之后，修复，部分使用标记为您感兴趣的组织在原位杂交和免疫染色或执行。 </li></ol><img src="/files/ftp_upload/1932/1932fig1.jpg" alt="图1" /> 图1。有机玻璃设计铸造橡胶模具，用于胚胎控股菜。一个38 x 38毫米圆角方形（〜5毫米深）已经排到了一块50 × 50平方毫米和25毫米厚的有机玻璃。圆形，均匀分布的井直径（1.5毫米）已钻深1毫米到塑料。为了使弹性体的显微注射菜模具，倒入有机玻璃Sylgard弹性，使得它能够巩固，按制造商的说明。这弹性模是用来注射和第隔离板。 <img src="/files/ftp_upload/1932/1932fig2.jpg" alt="图2" /> 图2。排序前移植的动物帽是重要的，因为表达可以有所不同 。动物在左边的第表示mCherry整个组织均匀，而右边的外植体参差不齐的表达，都将被丢弃。 <img src="/files/ftp_upload/1932/1932fig3.jpg" alt="图3" /> 图3。主机tadpOLE开发了一个眼状结构，其侧翼从移植的动物第细胞。一种荧光图像已明的形象重叠。

<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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<tr>
<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 ...

tissue-determination-using-animal-cap-transplant-act-assay-xenopus

Research

JoVE Journal

Biology

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

12.1K 次观看.  SUNY Upstate Medical University. 动物帽高表达的基因产物（S）移植到发展中国家的侧翼非洲爪蟾胚胎，以建立组织是否确定。

视频: 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

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

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

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

Obtaining Eggs from Xenopus laevis Females

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, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

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, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

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

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

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

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 ...

Mammary Epithelial Transplant Procedure

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, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

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, ...

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 ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

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

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

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

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

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 ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...