这项工作是由德国研究基金会（DFG; LU1455/1-1）的资助支持。

1。 前大毛文化<ol><li>在强制通风孵化器（BSS160，Ehret，德国）在37.5°C间新鲜受精卵奠定孵化湿度60％到他们的长边。在12°C储存时间超过一个星期的鸡蛋不应使用。 </li><li> 2.5天（约阶段HH17）孵化后，取出鸡蛋和标签顶端，用铅笔表明开裂方向。 </li><li>在打击之前，倒入一个干净的大培养皿（直径145毫米;格雷纳生物公司之一，德国），约20毫升无菌蒸馏水。 </li><li>到大培养皿中，将另一个小的无菌培养皿（直径94毫米;​​格​​雷纳生物一公司）。 </li><li>对锋利的金属边缘的底部裂缝的鸡蛋，小心翼翼地打开鸡蛋和小培养皿转移到每个鸡蛋的整个内容。 </li><li>大培养皿盖上盖，投入另一个孵化器（粘合剂有限公司，Tuttlinge，德国）为进一步培养在37.5℃，湿度60％左右。胚胎孵化所需的天数后，可用于电。 </li></ol> 2。 前OVO电穿孔的制备<ol><li>拉玻璃毛细管与电极拔（普勒100微管拖轮;世界精密仪器，柏林，德国）使用玻璃管直径1.0毫米（TW100F-4;世界精密仪器），并打破玻璃毛细管到一个合适的一角直径。 </li><li>设置适当的参数（即，为不同组织和胚胎的大小不同的电压）为电和电极连接到一个electroporator（编辑; CUY21-NEPA基因，千叶，日本），根据靶组织和胚胎的大小。 </li><li>准备含有质粒pCAGGS载体靶基因编码，例如，cadherin7（Cad7; pCAGGS-Cad7浓度为2.0微克/微升）和标记基因质粒的解决方案，例如，绿色荧光蛋白（GFP; pCAGGS-GFP的浓度为0.25微克/微升）。再加入终浓度为0.1％（Sigma公司）绿色标签质粒的解决方案的快速绿色。 </li><li>加载玻璃毛细管质粒用口吸管的解决方案。 </li></ol> 3。由前大毛电穿孔基因转移到鸡顶盖<ol><li>经过前大毛文化，例如，通过孵化日（五）6，鸡胚胎用于前大毛电。 </li><li>仔细在顶盖撕裂卵黄和羊膜膜用细镊子和顶盖表面添加无菌0.9％氯化钠溶液3滴。 </li><li>由玻璃注入质粒的解决方案，用口吸管毛细血管到顶盖腔。 </li><li>放置电极，钨针阴极和旁边顶盖矩形板阳极（CUY 661-3x7，NEPA基因），立即申请的顶盖electroporator所产生的电脉冲（6脉冲，25V，60毫秒脉冲长度，每个案件100毫秒间隔）。 </li><li>覆盖的大型培养皿的盖子，并返回进入孵化器。适当天（例如，电击后2天或3天）的潜伏期后，顶盖的收集和固定染色（ 图1）和生物检测。 </li></ol> 4。代表结果成功由前大毛电Cad7和绿色荧光蛋白的外源蛋白的过度表达作为一个例子，在图1所示。当Cad7与GFP质粒质粒一起到顶盖电穿孔的E6电后，两三天内，胚胎收集和固定。在E8的，强烈表达绿色荧光蛋白在整个安装图像（ 图1A-C），顶盖。此外，在九个人口众多的顶盖部分，绿色荧光蛋白（ 图1D绿色）和Cad7蛋白（ 图1E红色）共表达（ 图1F黄色），这表明前大毛电是一个成功的基因转移到鸡胚胎在体内的旧方法。 <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="图1"/> 图1。当质粒编码绿色荧光蛋白和Cad7的E6转染鸡顶盖， 前大毛电后两三天，绿色荧光蛋白（绿色）强烈表达（交流）wholemount的图像显示在E8的。在九个人口众多的顶盖部分，绿色荧光蛋白（D中的绿色）和Cad7蛋白（红色在E）共表达（在F黄色）。高炉，亮场图像。比例尺:在一个200微米交流;为DF在D 100微米。

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., 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=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</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.">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

没有利益冲突的声明。

该协议提供了旧鸡胚前OVO电（例如，将在E6顶盖）的转基因指南。这种方法延长使用电老鸡胚胎，并在稍后阶段研究基因在体内的功能可以方便地应用于许多实验室。至少从E4类E7的胚胎可以用这种方法成功的电穿孔和电穿孔胚胎可以存活直到E15 11。除了 ​​大脑，这种方法还可以用于基因转移成鸡肢体制度11。 应特别注意支付给由前OVO电?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> electroporator </td><td>日本NEPA基因， </td><td> CUY21的编辑</td></tr><tr><td>电极</td><td>日本NEPA基因， </td><td> CUY661-3x7 </td></tr><tr><td>鸡蛋孵化器</td><td> ehret，德国</td><td> BSS160 </td></tr><tr><td>胚胎孵化器</td><td>粘合剂有限公司，德国</td><td></td></tr><tr><td>荧光显微镜</td><td> KEYENCE，德国公司</td><td> BZ-8000 </td></tr><tr><td>培养皿</td><td>格雷纳生物，其中有限责任公司，德国</td><td></td></tr><tr><td>微电极拔</td><td>世界精密仪器，德国</td><td>的Pul-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

鸡胚胎为研究胚胎发育过程中基因功能和调控提供了一个很好的模型系统。 在OVO电是一个强大的过度表达外源基因的方法或下调体内的内源性基因在鸡胚1。不同的结构，如DNA质粒编码基因的小干扰RNA（siRNA）的质粒5，小合成的RNA寡核苷酸6，和吗啉反义寡核苷酸7 2-4，可以很容易地通过电转染鸡胚胎。然而， 在OVO电的应用是有限的胚胎早期孵化阶段（年龄小于阶段HH20 -据汉堡和汉密尔顿）8，有一些缺点是其在胚胎中的应用，在稍后阶段（比HH22阶段老年人-约3.5发展天）。例如，在稍后阶段卵黄膜是通常盟友坚持应当膜，并在外壳打开一个窗口，导致血管破裂，导致胚胎死亡;年纪较大的胚胎卵黄囊和尿囊血管，它是很难访问和操纵胚胎覆盖旧胚胎移动大力通过在shell中比较小的窗口，是难以控制的方向。 在这个协议中，我们证明前OVO电穿孔法基因转移到鸡胚胎后期（比HH22阶段旧）。对于前大毛电，是胚胎培养在培养皿9和卵黄和尿囊血管被广泛流传。在这些条件下，年龄较大的鸡胚胎很容易访问和操纵。因此，这种方法克服缺点，适用于年龄较大的鸡胚胎的OVO电。质粒使用这种方法，可以很容易地转成不同的部分老鸡胚胎10-12。

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

介绍将在以后的孵化阶段（比汉堡和汉密尔顿阶段（HH）22岁以上）的鸡胚胎的基因转移方法。这种方法克服缺点<em&gt;在OVO</em&gt;电适用于年龄较大的鸡胚胎是一种有用的技术，研究基因功能和调控，在旧的发展阶段。

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K 次观看.  School of Medicine University of Rostock. 介绍将在以后的孵化阶段（比汉堡和汉密尔顿阶段（HH）22岁以上）的鸡胚胎的基因转移方法。这种方法克服缺点在OVO电适用于年龄较大的鸡胚胎是一种有用的技术，研究基因功能和调控，在旧的发展阶段。

视频: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...