Name: optimized ex-ovo culturing of chick embryos to advanced stages of development
Uploaded: 2015-01-24T14:00:00
Description: Watch this Scientific Journal Video about Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development at JoVE.com

我们要感谢保罗·普瓦里耶，媒体制作，在圣文森特山大学为他的拍摄工作和编辑这篇稿子的视频部分。我们承认自然科学加拿大与工程研究理事会资助。

注：所有用品都列在表1中。 1.存放鸡胚<ol><li>孵育应变鸡水平鸡在37℃，大约40％的湿度的鸡蛋，把鸡蛋一次或每天两次。翻蛋重要的是要防止胚胎附着在蛋壳。 </li><li>不打开鸡蛋在24小时之前，建立培养另有胚胎将位于腹侧蛋黄的质量和在打开鸡蛋在步骤3除了将被损坏，保持蛋在4℃下度孵化，以“叫停”开发前不超过一个星期，但这种不理?...

<ol>
	<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. 41, 391-394 (1974).</li><li>Gennaro, L. D., Packard, D. S., Stach, R. W., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+chicken+embryos+in+simplified+shell-less+cultures+under+ordinary+conditions+of+incubation.">Growth and differentiation of chicken embryos in simplified shell-less cultures under ordinary conditions of incubation.</a> Growth. 44, 343-354 (1980).</li><li>Tufan, C. A., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanat. 3, 8-11 (2004).</li><li>Duench, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+and+Hedgehog+signaling+during+the+development+of+scleral+ossicles.">BMP and Hedgehog signaling during the development of scleral ossicles.</a> Dev. Biol. 365 (1), 251-258 (2012).</li><li>Datar, S., Bhonde, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+Chick+Embryo+Culture+as+an+Alternative+in+vitro+Model+to+Investigate+Glucose-Induced+Malformation+in+Mammalian+Embryos.">Shell-less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformation in Mammalian Embryos.</a> Rev Diabet Stud. 2 (4), 221-227 (2005).</li><li>Fisher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryos+in+shell-less+culture.+Tested+studies+for+laboratory+teaching.">Chick embryos in shell-less culture. Tested studies for laboratory teaching.</a> , (1983).</li><li>Dunn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+shell-less+culture+of+the+72-hour+avian+embryo).">Technique for shell-less culture of the 72-hour avian embryo).</a> Poultry Science. 53, 409-412 (1974).</li><li>Yalcin, H., Shekhar, A., Rane, A. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Silver, P. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+problems+of+experimenting+in+ovo+on+the+early+chick+embryo,+and+a+solution.">Special problems of experimenting in ovo on the early chick embryo, and a solution.</a> J Embryol Exp Morph. 8 (4), 369-375 (1960).</li><li>Spurlin, J., Lwigale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations.">A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.</a> Dev. Dyn. 242 (2), 148-154 (2012).</li><li>Dorrell, M. I., et al. <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+model+for+directly+visualizing+chicken+embryo+development.">Ex ovo model for directly visualizing chicken embryo development.</a> American Biology Teacher. 74 (9), (2012).</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. Morph. 88, 49-92 (1951).</li><li>Drossopoulou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anteroposterior+patterning+of+the+vertebrate+limb+based+on+sequential+long-and+short-range+Shh+signaling+and+Bmp+signaling.">A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signaling and Bmp signaling.</a> Development. , 127-1337 (2000).</li><li>Sys, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J. Vis. Exp. (77), e50522 (2013).</li><li>Franz-Odendaal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in chicken, Gallus gallus.</a> Dev. Dyn. 237, 3240-3251 (2008).</li></ol>

前大毛培养和窗口都有优点和挑战。在这里，我们比较一下优点和泡沫塑料杯前卵内的方法和开窗的方法对我们的优化卵内的方法当然在这里显示的挑战。我们的方法能够操纵和便于观察小鸡胚胎在发育的后期阶段，我们的改进传统的前卵方法1，2，3，使其附加地很容易在本科教学实验室类使用。 虽然许多研究人员更加窗方法来研?...

Ex ovo culturing has played an important role in the study of development of the chicken1, 2. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes 3, 4, 5.
There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup6,7,8 or a glass bowl5. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity6. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses. 
The above challenges in the ex ovo method has meant that researchers turn to the windowing method 9,10 to view embryonic chick development. In this technique, a hole or &#8220;window&#8221; is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax9 to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 &#8211; HH 27), viewing the entire embryo at late stages is not easily accomplished. 
Here, we describe an improved and simple ex ovo culturing technique11 that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

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			<td height="78" style="height:79px;width:177px;">Penicillin/Streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:152px;">P4458</td>
			<td style="width:192px;">Make small aliquots to avoid freeze/thaw events</td>
		</tr>
		<tr height="22">
			<td height="61" rowspan="3" style="height:61px;width:177px;">Square Petri Dish</td>
			<td rowspan="3" style="width:136px;">N/A</td>
			<td rowspan="3" style="width:152px;">N/A</td>
			<td rowspan="3" style="width:192px;">9.5 cm x 9.5 cm</td>
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			<td height="22" style="height:22px;width:177px;">Weigh Boat</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td align="right" style="width:152px;">8732113</td>
			<td style="width:192px;">88 x 88 x 23 mm</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Ziplock container</td>
			<td style="width:136px;">Ziplock</td>
			<td style="width:152px;">N/A</td>
			<td style="width:192px;">12 cm x 12 cm x 6 cm</td>
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optimized ex-ovo culturing of chick embryos to advanced stages of development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

这当然大毛方法可以从开发的初期阶段（HH 19/20），观察胚胎的发展（ 图1B和1A），后期（HH 40-41）。建立文化的HH 19-20增加了文化的胚胎存活。在此之前的头部转动（前53 HPF）生存能力是很低的文化和级21之后，胚胎往往如此少完整胚获得更粘到上除去外壳。在一般情况下， 前OVO文化的胚胎生存能力更是高达35-36 HH（90-100％），但它确实下降的发展更高级阶?...

Watch this Scientific Journal Video about Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development at JoVE.com

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Research

JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â A High-throughput Screen in ES Cells

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Isolation of Preadipocytes from Broiler Chick Embryos

从肉鸡雏鸡胚胎中分离出前脂肪细胞

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...