这项工作是由国立卫生研究院拨款R01 GM075200和R01 HL083393（LAT）的支持。我们承认从NIH T90的DA022871和Mallinckrodt放射研究所曝气生物滤池的奖学金支持，并从美国心脏协会09PRE2060795授予VDV值。

1。一般实验准备<ol><li>准备在层流罩组织培养基。 <ol><li>稀贝科改良Eagle培养基（DMEM）（1大号瓶4.5克/ L葡萄糖，碳酸氢钠，和L -谷氨酰胺）。加入10 mL青霉素/链霉素/新霉素抗生素。 </li><li>取出用无菌移液管的DMEM 100毫升和100毫升鸡血清取代。 </li><li>分装DMEM/10％的雏鸡血清/ 1％抗生素无菌的15 mL锥形瓶和冻结。 </li></ol></li><li>准备磷酸盐缓冲液（PBS）补充钙和镁。混合100毫升10X PBS中，去离子水900毫升，集中解决钙和镁（100毫克/毫升氯化钙2•2H 2 O，100mg/mL氯化镁2•6H 2 O）和1毫升。 </li><li>在汉堡和汉密尔顿1（即23-25 ​​小时HH阶段5，或42定义所需的阶段，作为一个纵向方向孵育鸡蛋HH阶段11-12）-48小时。 </li><li>取出胚胎的鸡蛋使用的滤纸载体法。 <ol><li> A150毫米培养皿侧面裂纹鸡蛋的底部。从底部拉开壳和空入菜蛋的内容。 </li><li>从蛋的钝钳取出较厚，粘性蛋白。 </li><li>切的Whatman＃2滤纸2-3厘米的圆圈。直径。在滤纸中使用单一打孔，打孔。蛋黄的方式，使胚胎可见通过在环中心孔的滤纸环广场之一。 </li><li>切周围的外径与microscissors滤纸。从蛋黄用细镊子取出纸张。 </li><li>胚胎在PBS轻轻冲洗，以清除附着卵黄颗粒。 （HH5胚胎可能需要长时间浸泡15-20分钟以清除残留的蛋黄）。从浴室取出滤纸组件放置在腹侧35毫米培养皿。将第二个过滤器上盖的第一个纸环，夹环之间的胚胎。周围放上滤纸三明治（≈1.5 - 2厘米直径）的外部金属环，以确保胚胎。 </li><li>淹没在培养基中的胚胎。验证正确的形态与明场显微镜和萌芽阶段。 </li></ol></li><li>使用移液管拉拔尖端细的玻璃针（1.5毫米内径）（世界精密仪器，萨拉索塔佛罗里达州），准备随后组织解剖和标签。 </li></ol> 2。 DII标签5 HH阶段的胚胎使用磁性铁粒子<ol><li>添加少量的铁粉（铁减少，Mallinckrodt贝克公司，Phillipsburg，NJ）在室温饱和DII在几滴酒精，让干。 （这通常会导致颗粒蛋糕，一旦酒精蒸发。）使用一把拉住微量的一角，打破了成群的铁粒子，他们转移到离心管，冲洗数次，并盖上去离子水。 </li><li>标签了... ...第5阶段的胚胎外胚层细胞，收获的胚胎背侧（即整个滤纸大会）在35毫米的文化与文化传媒的慷慨层覆盖的菜，并在解剖显微镜下的菜。使用玻璃针去除卵黄膜和揭露外胚层细胞。 （小心不是皮尔斯胚胎。） </li><li>绘制一个列标记的铁粒子进入巴斯德吸管（去离子水）。使用解剖显微镜，淹没枪头下方的液体表面以上的胚胎的位置。吸管慢慢地来回滚动之间的指尖，争相粒子的pipiette洒在整个胚胎。 </li><li>一旦胚胎布满颗粒，小心地放置在37℃培养箱中，约10分钟。利用胚胎孵化器了D使用一个强有力的磁铁，以消除粒子。 </li><li>使用连接的摄像头和适当的过滤器设置荧光显微镜获得的胚胎明场和荧光图像，在图2A，B所示</li><li>注意标签的密度。如果所需的高密度分布，使用额外的颗粒（如上所述）标签胚胎，并相应延长孵化时间。如果标签过于密集，添加少量的离心管中填充粒子与已标记和未标记的铁粒子混合管内容。重复标记过程与一个新的胚胎，并再次注意标签密度。 </li></ol> 3。光学相干断层扫描（OCT）成像聚苯乙烯微球标记HH11 - 12胚胎<ol><li>准备在35毫米培养皿的黑10微米的微球直径（Polysciences公司，灵顿，PA）在PBS的解决方案。 （珠应足够大，产生华侨城的对比度成像，但足够小的如珠大小的单个细胞的顺序是。具体数值将取决于您的OCT系统的决议，并正在研究的组织）。一滴原液每毫升的PBS（2.6％固体乳胶）就足够了。拉成10CC使用22号针头的注射器这一解决方案。涡的解决方案为30秒，以确保珠均匀分布。 </li><li>锥拉玻璃针使用的计算机硬盘驱动器2的提示。提示直径应足够大，珠可容纳通过开放，但在同一时间，尽可能小，以尽量减少注射过程中的组织伤人。 </li><li>填写珠使用注射器的解决方案的斜角玻璃针。轻轻一抖，以确保该解决方案已达到提示的微量。 </li><li>利用显微操作，胚胎放置在同一领域的玻璃针。珠应流淌出来的枪头。 <ol><li>如果没有BEADS退出的微管，有可能被堵塞。在这种情况下，重复步骤一个新的玻璃针3.2-3.4。 </li><li>如果有太多的珠云您的视野，你的枪头可能太宽。一个新的吸管和一个新的胚胎的重复步骤3.2-3.4。 </li></ol></li><li>枪头插入大脑管的管腔。要确保没有捅破组织腹楼。你会看到一个组织的瞬态肿胀如珠和液体进入管腔。这表明一个成功的插管;现在，迅速取出玻璃针。 </li><li>验证在使用明场显微镜的大脑管的内腔足够珠密度。让珠为20 - 30分钟解决节之前。 4。 </li></ol> 4。数据采集 *下面的方法非常适合于以上所述的荧光标记技术。然而，随着微球标记技术，珠子可以打跑转移样品时孵化器和成像系统。在这种情况下，最好进行步骤4.2（运动/搅拌完全避免样品）。在这两种方法，胚胎，而淹没在液体培养基中培养。如果胚胎没有完全淹没（这是一些传统的组织培养技术的情况下），组织几何大大高得离谱的表面张力负载和应变分布改变，将无法准确地捕捉正常的3 - D形态发生的3，4。此外，淹没组织可以有效地防止了鲜明的对比观察OCT成像在液气界面模糊胚胎形态和标记坐标。 <ol><li>胚胎培养（一般） <ol><li>您的第一张图像（明场，荧光，或华侨城）收购后，把700 35毫米培养皿（胚胎），在150毫米的培养皿中，并放置在一个小塑料袋整个装配。添加夫妇滴去离子水的加湿袋95％O 2 / 5％CO 2混合，并填写收入囊中。胚胎放入孵化器在37 ° C（队列Stabiletherm，阿什维尔，北卡罗来纳州），直到下一次成像时间点所需的时间。使用这种方法，可以进行实验在多个胚胎，在同一天。 </li></ol></li><li>时间推移文化（自动化） <ol><li>含有1.2毫升的培养基放入一个三角洲T盘（外径35毫米，23毫米圆锥侧壁的中央孔径，0.17毫米厚的玻璃; Bioptechs，巴特勒，PA）标记的胚胎。 </li><li>盖菜，保持了玻璃盖在37℃用一个Bioptechs三角洲T4的培养皿控制器（防止结露）。 Superfuse与95％O 2 / 5％CO 2混合气体从一个微型泵变流量装置（尔科技）提供的胚胎。之前到达胚胎，热和加湿温暖（&lt;100 ° C）热板通过去离子水冒泡的气体。本实验的S示意图等，如图1所示。 </li><li>成立了软件自动获取在用户指定的时间间隔图像。对于我们的荧光显微镜下，我们使用的OpenLAB软件（珀金埃尔默，马萨诸塞州沃尔瑟姆），并为我们的OCT系统，我们使用席卷源Thorlabs软件（牛顿，新泽西州），由于此方法是目前唯一能够处理一个时间推移实验一时间，它往往是最好的，让这些类型的文化运行过夜。通过安装一个自动翻译的成像系统的硬件和软件兼容的阶段，人们可以绕过这个限制。 </li></ol></li></ol> 5。代表性的成果: 标签自动​​跟踪（使用Volocity，珀金埃尔默）或手动（使用ImageJ手动跟踪插件，NIH）的每一个实验。在荧光标记技术，我们使用Matlab的常规gridfit适合通过标记坐标的二维表面，从而使<html>形成表面株计算6，7。标准方程是使用这些值转换成相关的胚胎坐标系。另外，在OCT技术，表面生成的分割图像卷（通过标准软件，如Matlab或插入符 8获得）和应变可以计算出样本 9的最大或最小曲率方向。 我们用我们的铁粒子技术标签和跟踪外胚层细胞在早期鸡胚在头褶皱形成的议案。如图2a所示，荧光标记的细胞，分布在整个胚胎。明场和荧光图像的胚胎被抓获，在不同的时间间隔期间前 OVO文化。跟踪标签的议案（图2B，C），然后用头褶皱形成的形态在不断变化的应变分布（图2D - F）的计算。 T"&gt;同样，我们的聚苯乙烯微球技术被用于跟踪在早期雏鸡脑组织中后脑边界运动。正如图3屋宇署所示，珠议案追踪6小时和应变特征在纵向和组织变形圆周方向分别计算标记的坐标。注意，此方法能够处理明显的3 - D变形珠往往坚持各方脑的内腔（图3A）。 <img src="/files/ftp_upload/3129/3129fig1.jpg" alt="图1"/> 图1设置时间推移组织培养的原理。 <img src="/files/ftp_upload/3129/3129fig2.jpg" alt="图2"/> 图2。量化在使用荧光标签追踪早期的小鸡胚胎的2 - D组织变形。 （一）合并的明场/荧光图像的HH第5阶段的胚胎后去除铁粒子。 DII的标签分布在整个胚胎版外胚层细胞（红色）。 （B，C）标记细胞的运动跟踪使用ImageJ。标签位移计算胚胎坐标（X，Y，）。需要注意的是A和B是相同的图像。 （DF）的不断发展的过程中头部折叠形成纵向应变分布的等高线图。纵向拉格朗日株计算相对为hh（四）75分钟后的第5阶段（即A和B），（五）120分钟，和（F）165分钟的潜伏期。这些菌株的特征线元素原本面向沿Y轴的长度变化（在第5阶段的胚胎）。使用追踪标签的坐标计算形态株的进一步细节，可以发现在Filas等人（2007）6 Varner等人（2010） 7。请注意，C和F是相同的图像。比例尺= 500微米。 <img src="/files/ftp_upload/3129/3129fig3.jpg" alt="图3"/> 量化的3 - D组织ð 图3。在早期鸡胚脑eformations。 （一）大脑管的聚苯乙烯微球，坚持背（黑色箭头）和腹侧（白色箭头）双方很容易从周围组织中的横向交叉华侨城重建section.Ventral意见明显显示中期后脑边界附近的微球的位置（三）（二）HH12和后6小时的潜伏期（男:脑H:后脑）。珠几组概述突出组织的整体变形。 （四）（五ZZ）纵向和环（五θθ）从这些变形计算拉格朗日株。积极的纵向和负中期后脑边界圆周株反映轴向延长本地区和文化过程中的圆周缩短。计算复杂曲面的形态发生过程中的形态株的详情，可发现 Filas等人 （ 2008）9。比例尺= 200微米。

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没有利益冲突的声明。

前OVO文化早期鸡胚两种组织标签技术。首先使用通过铁磁性颗粒的亲脂性荧光染料，同时标签数百个细胞。然而，这种方法是目前不兼容光学相干断层扫描，荧光染料一般使用10月10日从周围组织的对比度很小。因此，我们使用聚苯乙烯微球标记时间推移华侨城分析组织的一种替代技术。这种技术产生的3 - D数据集，但必须小心不逐出组织的珠子。在适当的实验环境中使用这些方法?...

<table border="1"><table border="1"><tbody><tr><td> 材料/试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液 - 高血糖</td><td> Sigma - Aldrich公司</td><td> D5796 </td><td></td></tr><tr><td>青霉素/链霉素/新霉素</td><td> Sigma - Aldrich公司</td><td> P4083 </td><td></td></tr><tr><td>鸡血清</td><td> Invitrogen公司</td><td> 16110-082 </td><td></td></tr><tr><td>贝科的磷酸盐缓冲液</td><td> Sigma - Aldrich公司</td><td> D1408 </td><td> 10X </td></tr><tr><td>用Whatman＃2滤纸</td><td>瓦特曼</td><td> 1002 090 </td><td> 90毫米直径</td></tr><tr><td>玻璃微量移液器</td><td>世界精密德州仪器（WPI） </td><td> TW150 - 6 </td><td> 1.5毫米内层直径</td></tr><tr><td> DII </td><td> Invitrogen公司</td><td> D - 282 </td><td></td></tr><tr><td>铁减少</td><td> Mallinckrodt贝克公司</td><td> 5320 </td><td></td></tr><tr><td> 10微米的微球直径（黑色） </td><td> Polysciences公司</td><td> 24294 </td><td></td></tr><tr><td>三角洲T盘（时间的推移文化） </td><td> Bioptechs </td><td> 04200415B </td><td> 0.17毫米厚，黑色</td></tr><tr><td>三角洲T4培养皿控制器</td><td> Bioptechs </td><td> 0420-4-03 </td><td></td></tr><tr><td>微型泵变流量设备</td><td> Fisher Scientific则</td><td></td><td></td></tr></tbody></table>

tracking morphogenetic tissue deformations in the early chick embryo

胚胎上皮细胞进行复杂的变形（如弯曲，扭曲，折叠和拉伸），形成早期胚胎的原始器官。跟踪基准对这些细胞的表表面标记是一个估算，如生长，收缩和剪切形态数量以及建立的方法。然而，并非所有的表面标记技术容易适应传统的成像方式，具有不同的优势和局限性。在这里，我们描述了两种标记方法，并说明每种技术的效用。在第一种方法，荧光标记的数百个应用同时使用铁磁性颗粒的胚胎。然后，这些标签使用量的2 - D组织变形过程中的形态发生。在第二种方法中，聚苯乙烯微球用于非侵入性光学相干断层扫描（OCT）成像造影剂跟踪的3 - D的组织变形。这些技术已成功LY落实在我们的实验室studythe初头倍，心脏和大脑发育，物理机制，并应适应范围广的形态发生过程。

Watch this Scientific Journal Video about Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo at JoVE.com

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

本文介绍了表面的标签和<em&gt;前OVO</em&gt;在早期鸡胚组织培养。时间推移明场，荧光，和光学相干断层扫描成像技术适合。高时空分辨率的跟踪表面标签，使运动的数量，如在二维和三维计算的形态株（变形）。

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

tracking-morphogenetic-tissue-deformations-in-the-early-chick-embryo

Research

JoVE Journal

Biology

12.9K 次观看.  Washington University. 本文介绍了表面的标签和前OVO在早期鸡胚组织培养。时间推移明场，荧光，和光学相干断层扫描成像技术适合。高时空分辨率的跟踪表面标签，使运动的数量，如在二维和三维计算的形态株（变形）。

视频: Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...