我们要感谢他的高清摄像头（炮VIXIA HFS100）使用玛斯恩维尔先生。这项工作是支持的，一部分由来自NIH的“（EY018738），新泽西州的脊髓研究委员会（08-3074-可控硅 - E-0和10-3091-SCR-E 0），布希生物医学研究奖补助。

由于该协议的某些部分进行轻微修改先前在其他的朱庇特的视频和论文3所述，我们不会在这里详细讨论。 1。鸡蛋处理和针的制备<ol><li>鸡蛋可以储存在13℃左右的葡萄酒在凉爽长达1周。如果温度过高，胚胎开始正常发展，而较低的温度下会导致高死亡率。一旦你准备好孵化从酒柜中取出的鸡蛋和鸡蛋与较大的一端设置垂直向上。保持至少2个小时之前，他们在37.5℃孵化的鸡蛋，在室温。 </li><li>胚胎第4天，以获取胚胎在汉堡和汉密尔顿阶段22 4-5 96-100小时的孵化蛋。 </li><li>准备从拉玻璃毛细管微量针头和打破根据解剖微尖一个镊子得到一个直径约0.1微米和20毫米锥尖端张开的范围。较大技巧的针头刺入卵黄膜而小技巧有困难装载和交付的DNA溶液的困难。 </li><li>将0.1毫升汉密尔顿气密性注射器安装在显微操作针。使用注射器针Masterplex硅胶管的小块。由于连接是紧无需添加矿物油的密封附件。 </li><li>不等3-6微克/μl和0.2μl快绿了一块封口膜（0.025％）的混合浓度记者质粒DNA溶液2μL。绿色染料将有助于快速可视化的注射。慢慢地，加载的混合物针。 </li><li>摆脱卵黄膜的内层膜，蛋轻轻旋转约180°，并等待数分钟，然后将其旋转回原来的位置，并设置它的电。 </li><li>擦拭的FORCEPS和蛋壳的胚胎，以避免感染，用70％乙醇。 </li><li>做一个小孔，在上面一对AA尺寸钳空气电池的鸡蛋。小心打击蛋壳。取出小块的时间，使一个小窗口之一。而不触及卵黄膜的使用镊子小心取出内膜。 </li></ol> 2。注射和电<ol><li>为了防止损害大脑或心脏，定位针禁忌主要的血液进入眼内，指向喙血管束外侧。 </li><li>皮尔斯通过卵黄膜，巩膜，视网膜和玻璃体突然轻推针。如果通过眼睛的另一端穿过针应该没事，除非它破坏任何主要的血液静脉。 </li><li>慢慢在开幕式的边缘拉回来针，并把它放在眼球的外壁几乎相切。 </li><li>插入背板T针巩膜和视网膜之间的视网膜进入子空间。 </li><li>注入的DNA，直到你能想象灌装一侧眼球，推动创建一个隆起的视网膜向内的绿色解决方案。 </li><li>如果你的针配售是不正确的，那么你将看到的DNA溶液，填补了中间的眼球内的玻璃体幽默的蔓延。另外，如果你损害视网膜太多，你会看到DNA溶液来眼球。 </li><li>慢慢去除针电极在PBS浸泡后立即放置在蛋内的平行。向下推入羊水淹没在使注入的眼睛是位于电极之间的电极。避免接触电极放置任何大血管或心脏。负电极应该在注射侧，使DNA可以从子视网膜空间运朝正电极的视网膜。电5 15V脉冲为50毫秒，950毫秒的间隔porate视网膜。 </li><li>小心地取出电极和密封件透明胶带鸡蛋的窗口。 </li><li>标签和日期之前把它孵化注入卵子。通常情况下，它需要约3至5分钟即可完成整个电过程。 </li><li> GFP的表达，可以看作是早8小时后，电。然而，你可能会等待，直到胚胎收获前达到预期的阶段。 </li></ol> 3。代表结果在我们的研究中，我们使用各种质粒结构的研究，涉及视网膜细胞发育的基因表达调控。在这个视频pCAG-GFP（染控制）被用来追踪一个成功的注射和电。然而，任何与记者质粒结构基因（绿色荧光蛋白，RFP等）都可以使用。尽管GFP的表达可以看出，早8小时后元素ctroporation，我们通常开始收获鸡蛋（E6），第6天起。电穿孔视网膜解剖胚胎前嵌入和切片分析和荧光解剖显微镜下。通常情况下，报告基因的表达，可以看到至少在一季度的视网膜，成功后的电（图1）。进一步分析转染视网膜组织切片通过细胞形态清晰的可视化。免疫组化使用的细胞类型的特定标记（Brn3a，PAX6等）允许特定的细胞GFP的表达特性（图2）。 <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> 图1。注射和电穿孔成功电记者质粒阳性结果GFP表达。鸡胚胎视网膜在胚胎第4天，并在胚胎第6天收获。至少有25％的视网膜成功转（=顶视图，乙=底视图）。比例尺= 1毫米。 <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> 图2。表达GFP的特性，采用免疫组织化学视网膜细胞，绿色荧光蛋白表达E7的阶段视网膜组织固定和切片。这些部分，然后沾上各种细胞特异性标记。 brn3a被用来确定神经节细胞（一），而PAX6是用来确定水平，无长突细胞和神经节细胞（B）。比例尺= 50微米。

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

所有的动物实验通过动物保健制度和设施委员会在罗格斯大学。

有针对性的视网膜注射，并在 E4类阶段OVO在胚胎视网膜电针对视网膜祖细胞，导致在单细胞水平上的所有六个主要的视网膜细胞类型的能力，可视化。 在OVO电在HH10（E1.5）针对视泡是能够转染细胞的发展，形成了眼。然而，这些细胞具有非常高的营业额，在这个时候，这种方法是不特定的视网膜细胞。这可能是高的细胞周转率，防止持续稳定表达。由E4时，胚胎不够发达，眼?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>受精卵无病原体（SPF）的白来航鸡蛋</td><td>朝阳农场（卡茨基尔，纽约） </td><td></td><td></td></tr><tr><td>鸡蛋孵化器</td><td> GQF制造，大草原，GA </td><td>型号-1550 </td><td>设置湿度60％和37.5°C。 </td></tr><tr><td>玻璃毛细管</td><td>世界精密仪器公司</td><td> TW150F-4 </td><td></td></tr><tr><td> 0.1毫升注射器</td><td>汉密尔顿有限公司，内华达州里诺</td><td>气密1710 </td><td>另外1ml注射器，以及可用于</td></tr><tr><td>硅的Masterflex（双氧水）管</td><td>科尔 - 帕默</td><td> HV-96400-13 </td&gt; <td>附加玻璃针注射器剪下一小块（1厘米） </td></tr><tr><td>镊子</td><td>杜蒙</td><td>机管局</td><td></td></tr><tr><td>微操作机器人</td><td>世界精密仪器公司</td><td> M3301-M3 </td><td></td></tr><tr><td>质粒DNA </td><td></td><td></td><td> pCAG-GFP是借来的，从康妮Cepko博士</td></tr><tr><td>固绿FCF </td><td> Sigma-Aldrich公司</td><td> F7252 </td><td>稀释用PBS为0.025％ </td></tr><tr><td>脉冲发生器</td><td>哈佛仪器，马</td><td> A型肉毒毒素流脑830 </td><td>方波发生器</td></tr><tr><td>电极</td><td>哈佛仪器，马</td><td> A型肉毒毒素模型514 </td><td>我们的电极相距3-5毫米间距</td></tr><tr><td>单色数码相机</td><td>德国蔡司， </td><td>一xiocam的MRM </td><td></td></tr><tr><td>荧光解剖显微镜</td><td>徕卡公司，德国</td><td>徕卡MZ16FA </td><td></td></tr><tr><td>直立荧光显微镜</td><td>德国蔡司， </td><td>蔡司Axio上成像仪A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

鸡胚胎的视网膜是一个很好的工具来研究在高等脊椎动物的视网膜发育。由于庞大的规模和外部的发展，它是相对非常容易操纵的鸡胚视网膜，利用重组DNA / RNA技术。胚胎视网膜电DNA / RNA的结构将有一个很大的优势，研究视网膜发育过程中视网膜干/祖细胞的基因调控。如检测报告基因检测，基因过度表达，基因敲除（shRNA的）等不同类型，可以使用电穿孔技术。该视频演示了有针对性的视网膜注射到鸡胚视网膜OVO在汉堡和汉密尔顿阶段22-23，这是关于胚胎第4天（E4），电。在这里，我们表明在OVO电技术的快速和便捷，使质粒DNA的表达绿色荧光蛋白（GFP）作为标记直接传递到鸡胚视网膜下的空间和通过电脉冲，以促进视网膜干/祖细胞的DNA吸收。视网膜注射和电E4类的新方法，让所有的视网膜细胞类型的可视化，包括后期新生神经元，这一直是注射和电的传统方法难以在E1.5 2。

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

该视频的总体目标是显示如何执行有针对性的视网膜注射<em&gt;在OVO</em&gt; DNA / RNA的结构，在汉堡包和汉密尔顿22-23阶段，这是关于胚胎第4天（E4），成鸡胚胎视网膜电。这种技术是非常有用的，以研究基因表达，基因调控，并在发展中国家小鸡视网膜的形态学变化。

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 ...

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K 次观看.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. 该视频的总体目标是显示如何执行有针对性的视网膜注射在OVO DNA / RNA的结构，在汉堡包和汉密尔顿22-23阶段，这是关于胚胎第4天（E4），成鸡胚胎视网膜电。这种技术是非常有用的，以研究基因表达，基因调控，并在发展中国家小鸡视网膜的形态学变化。

视频: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

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

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

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

小鸡前OVO文化前OVO CAM含量：它是如何真正起作用

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...