Biz onun filme iş ve bu yazının video bölümünü düzenlemek için Mount Saint Vincent Üniversitesi Paul Poirier, Medya Yapımcı, teşekkür etmek istiyorum. Biz finansman için Kanada Doğal Bilim ve Mühendislik Araştırma Konseyi kabul.

Not: Tüm malzemeleri Tablo 1&#39;de verilmiştir. 1. Tavuk Embriyolar Saklama <ol><li> Yaklaşık% 40 nem ile Gallus 37 o C yatay Gallus gerginlik tavuk yumurta inkübe ve bir kez ya da günde iki kez yumurta açın. Yumurta kabuğu Torna yapışmasını embriyo önlemek için önemlidir. </li><li> 4 o C&#39;de yumurta tutmak, önceki aksi embriyo sarısı kitle ventral yer alacak ve ek olarak 3. adımda yumurta açılması zarar görür gibi kültür kurma 24 saat içinde yumurta açmak etmeyin &quot;halt&quot; kalkınma inkübasyon öncesinde en fazla bir hafta derece ama bu ideal değildir. </li></ol> 2. Tavuk Embriyolar Sahneleme <ol><li> Hamburger ve Hamilton 12 evreleme tablosunu kullanarak tavuk embriyoları Sahne. Eski-ovo kültürlenmesini kurmak için ideal bir sahne HH evre 19-20 (inkübasyon 3- etrafında gün) bec olduğunuause Bu kafa 53 hpf döner kısa bir süre sonra olduğunu. Not: HH evre 19-20 aşağıdaki morfolojik özellikleri ile karakterize edilir: Somitlerin kuyruk çoğunluğu içine uzatılır, ancak kuyruk çok uç kuyruk tomurcuk kıvrılmış, bölünmemiş kalır, allantois küçük ve damarsal sınırlıdır, bacak-tomurcuklar kanat-tomurcuklar daha büyük ve gözler pigmentlenmemiş veya grimsi bir renk var. </li></ol> 3. Shell Embriyo Çıkarma <ol><li> Kabuktan embriyo çıkarmadan önce,% 70 etanol ile kabuk sprey ve kurumaya bırakın. Bu embriyo zarar verir gibi hızla yumurta açmayın. </li><li> Dikkatli olmak dikkatli alt tarafında yumurta çatlak, yumurta yönünü değiştirmek için değil (yani, inkübasyon sırasında ventral oldu tarafı) ve steril içine embriyo serbest tekne (88 x 88 x 23 mm) tartın. Silin% 70 etanol ile tekneler tartın. Yumurta sarısı çuval zarar görmediğinden emin olmak için kontrol edin embriyo. Yumurta sarısı h Eğerkırık gibi hayatta olmayacak gibi, embriyo atın. </li><li> O canlı olmasını sağlamak için embriyo uyun; Kalp atıyor, damar normal görünür, ve hiçbir belirgin anormallikler mevcuttur. Tartmak tekne kenarları kuru olduğundan emin olun. </li><li> Bir pipet kullanarak, enfeksiyonu önlemek için albümin üzerine penisilin / streptomisin, 40 ul (5.000 birim penisilin, ml başına 5 mg streptomisin) bırakın. </li></ol> 4. Nem Odası Hazırlama <ol><li> Kim-mendil ve / veya bir steril plastik konteynerin (12 x 12 x 6 cm ebadında), alt pamuk emici bir ped küçük bir deste koyun,% 70 etanol ile silinmiştir. </li><li> Kim-mendil ve / veya pamuk su (yaklaşık 150 mi) biraraya getirildi steril damıtılmış su ilave edin. </li></ol> 5. Ex Ovo Kültür Montaj <ol><li> Nemli dolgu üstüne embriyo içeren tartmak tekne yerleştirin. Ardından, t üstünde bir kare steril petri kabına (9.5 x 9.5 cm) yeri yarısıO gevşek kapak oluşturmak için tekne tartın. </li><li> Onun kapaklı plastik kap örtün. Hala odasının içine iyi hava akışı sağlar kısmi mühür sağlanması, kapağın iki köşeleri aşağıya doğru bastırın. </li><li> Dikkatle istenilen aşamaya kadar 37 o C inkübatör içine kurulum yerleştirin. </li><li> Kontrollü nem inkübatör kullanılamaz ise, nem kontrolü yardımcı inkübatör su kapları yerleştirin. Nem odalarında ve inkübatör tüm suyu sterilize ve kuluçka döneminde gerektiği gibi doldurun. % 40 nem ideal. </li></ol>

<ol><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">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/pubmed?term=((Growth+and+differentiation+of+chicken+embryos+in+simplified+shell-less+cultures+under+ordinary+conditions+of+incubation%5BTitle%5D)+AND+%22Growth%22%5BJournal%5D)">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/pubmed?term=((Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology%5BTitle%5D)+AND+%22Neuroanat%22%5BJournal%5D)">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/pubmed?term=((BMP+and+Hedgehog+signaling+during+the+development+of+scleral+ossicles%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">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/pubmed?term=((Shell-less+Chick+Embryo+Culture+as+an+Alternative+in+vitro+Model+to+Investigate+Glucose-Induced+Malformation+in+Mammalian+Embryos%5BTitle%5D)+AND+%22Rev+Diabet+Stud%22%5BJournal%5D)">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>Chick embryos in shell-less culture. Tested studies for laboratory teaching. Fisher, C. J. 5th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), , (1983).</li>
<li>Dunn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technique+for+shell-less+culture+of+the+72-hour+avian+embryo%29%5BTitle%5D)+AND+%22Poultry+Science%22%5BJournal%5D)">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/pubmed?term=((An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Special+problems+of+experimenting+in+ovo+on+the+early+chick+embryo%2C+and+a+solution%5BTitle%5D)+AND+%22J+Embryol+Exp+Morph%22%5BJournal%5D)">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/pubmed?term=((A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">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/pubmed?term=((Ex+ovo+model+for+directly+visualizing+chicken+embryo+development%5BTitle%5D)+AND+%22American+Biology+Teacher%22%5BJournal%5D)">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/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22J.+Morph%22%5BJournal%5D)">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/pubmed?term=((A+model+for+anteroposterior+patterning+of+the+vertebrate+limb+based+on+sequential+long-and+short-range+Shh+signaling+and+Bmp+signaling%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Towards+understanding+the+development+of+scleral+ossicles+in+chicken%2C+Gallus+gallus%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Towards understanding the development of scleral ossicles in chicken, Gallus gallus.</a> Dev. Dyn. 237, 3240-3251 (2008).</li>
</ol>

Yazarlar bu yazının sunulan bilgilere açısından hiçbir rakip mali çıkarlarını var.

Kültürleme ve her ikisi de pencereleme ex ovo avantajları ve zorlukları vardır. Burada avantaj ve yöntem ovo Strafor bardak ex ve burada gösterilen yönteme ovo Optimize ex pencere yöntemi zorlukları karşılaştırın. Bizim yöntem geç gelişim evrelerinde ve yöntem 1, 2 ovo geleneksel ex bizim ayrıntılandırmaları de manipülasyon ve civciv embriyo rahatlıkla izlenebilmektedir, 3 lisans öğretim laboratuvar derslerinde kullanmak üzere, ek çok kolay. Bunu gerçekleştirmek için basit ve gelişim geç evrelerinde (HH 41-42) mükemmel beka çünkü birçok araştırmacı tavuk gelişimini incelemek için pencere yöntemi tercih olsa da, sınırlamaları vardır. hangi büyük aşamasından sonra HH HH 30 evre 30 sonra tüm embriyo görüntülemek için sınırlı yeteneği, yumurta kabuğu pencere büyüyen, hareketli embriyo görmek için çok büyük olması gerekir. Bu büyük pencere fark olduğunuicult tamamen (kısırlık ve beka ile sorunlara yol açan), ve zor olabilir yumurta içinde iyi aydınlatma elde mühür. Buna ek olarak, ileri evre embriyolar pencereden büyük ve ulaşılması zor (veya işlemek) vardır. Burada tarif edilen yöntemle ex ovo embriyo tam sınır tanımayan erişim sağlar. Ağırlık tekne nem odasına yerleştirilir sonra, tamamı için çok kararlıdır. tün nem odası bir mikroskop altında yerleştirilebilir ve böylece yüksek büyütme tüm embriyo gözlemlemek için kullanılabilir. Buna karşılık, Strafor kap yöntemi uzun boylu bir nem odası (fincan yükseklik) sahiptir ve bu çok zor mikroskop hedefleri altına yerleştirmek için yapar. fincan tasarımı tekne tartmak da çok daha az kararlı düz daha ve bu bardak kolayca devirir olabilir. Yönteme ex ovo Bizim iyileştirmeler bir sınıf ya da laboratuar ortamında kullanmak için bu yöntem basit hale ve st sağlarHH 40 kadar sahnede HH 19 embriyolar tam erişim için udent; büyük organogenezisin oluştuğu zaman aşamaları. yöntemin ovo bizim eski en büyük dezavantajı kısırlık olduğunu. Enfeksiyonu önlemek amacıyla, bizim yöntem antibiyotik eklenmesini içerir. Antibiyotik bir doz daha sonraki aşamalarında embriyo verilebilir. Kültür set-up sırasında uygulanan antibiyotikler de embriyonun gelişimini etkilediği görünmüyor. Dramatik beka artırabilir kullanmak için önce% 70 etanol ile kapları sterilize. Genel olarak, yöntemin ex ovo gelişimi erişim veya sahnede kısıtlaması olmaksızın embriyolar görüntüleme ve işlenmesi için idealdir. Önceki araştırmalar embriyolar ex ovo kültürleme olumsuz 4,15 desenlendirme etkilemez göstermiştir. Örneğin, biz yumurta kabuğu olmamasına rağmen desenleme veya iskeletin indüksiyon herhangi bir değişiklik görmüyorum. Bizim gelişmiş yöntem birden c çözeryöntemiyle 1,2, 3 ovo mevcut geleneksel eski ile mevcut hallenges.

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.

<table><tbody><tr><td>Penicillin/Streptomycin</td><td>Sigma</td><td>P4458</td><td>Make small aliquots to avoid freeze/thaw events</td></tr><tr><td>Square Petri Dish</td><td></td><td></td><td>9.5 cm x 9.5 cm</td></tr><tr><td>Weigh Boat</td><td>Fischer Scientific</td><td>8732113</td><td>88 x 88 x 23 mm</td></tr><tr><td>Ziplock container</td><td>Ziplock</td><td>N/A</td><td>12 cm x 12 cm x 6 cm</td></tr></tbody></table>

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.

Yöntemin ovo Bu eski geç gelişim evrelerinde (HH 40-41) (Şekil 1A ve 1B) gelişme erken aşamalarında (HH 19/20) embriyoların gözlem için izin verir. HH 19-20 de kültür kurma kültür embriyoların beka artırır. Hayatta kalma kültürü ve sahnede 21 sonra çok düşüktür (53 hpf önce) çevirerek kafasına önce, embriyo daha az sağlam embriyolar elde edilir, böylece kaldırılması üzerine kabuk daha sopa eğilimindedir. (Yaklaşık% 40 HH 40-41 kadar hayatta) Genel olarak, kültür ex ovo embriyonun beka HH 35-36 (% 90-100) kadar yüksek, ama gelişme daha ileri aşamalarında düşüyor. Bu gelişme göstermiştir olmasına rağmen Ayrıca, bu geç evrelerde normal görünür ve iskeletin desenlendirme 4,15 etkilenmez, biz bacaklarda kemikleşme derecesi veya zamanlaması etkilenmiş olabileceğini düşündürmektedir bu son aşamada bükülmüş olduğunu gözlemledik. Buşaşırtıcı değil kemikleşme için kabuk kalsiyum ihtiyacını göz önünde bulundurarak. Kontrollere göre embriyonun gelişimi ayrıntılı bir karşılaştırma devam etmektedir ve bu yöntemler yazının kapsamı dışındadır. Gelişim boyunca embriyo erişim sağlamanın iki ana nedenden dolayı önemlidir. İlk olarak, araştırmacılar açmak ve bir pencere yeniden mühürlemek zorunda kalmadan günlük bazda gelişimini görmek mümkün. Örneğin, bireylerin birden fazla aşamada (Şekil 1C ve 1D) de uzuv ve göz gelişimini gözlemlemek mümkün. İkincisi, kültürleme ex ovo, embriyo daha (sınırsız) erişim sağlar ve böylece daha kolay manipülasyonlar veya ameliyatlar sağlayan. Embriyonik manipülasyonlar genellikle, bir stereomikroskop kullanılarak gerçekleştirilmesi gerekir. Strafor / plastik wrap yöntemine 6,7,8 aksine burada açıklanan kültür yöntemiyle ovo eski kullanılarak set-up kolayca und yerleştirilir embriyoer mikroskop, daha az derin ve devrilebilir olmaz. Embriyo engellemeyecek hiçbir engel (yumurta kabuğu) olmadığından ve oda çok kararlı olduğundan bu, gerekli ve embriyo erişimi optimize etmek döndürmek için tam nem odasını yerleştirmek için araştırmacı sağlar. Genel olarak, embriyo tartmak tekne aldığı destek nazik basınç set-up devrilmesini olmadan manipülasyon sırasında embriyo uygulanacak sağlar. Çeşitli gelişim biyolojisi yöntemleri bu kültür sisteminde embriyolara üzerinde yapılabilir. Bunlar detaylı organ gözlemler (örneğin, göz, beyin vb) veya teratojenik ajanlar doku gelişimini (örneğin, kan damarı büyümesi) veya uygulamayı içermektedir. Manipülasyonlar farklı doku katmanları birleştirerek veya tümör büyümesini ve metastazı 14 okuyan etkisini incelemek için koriyoallantoik membranlar üzerine dokuların greftleme içerir. Gelişimi sırasında yapılan başka popüler manipülasyon bea olduğunud veya bariyer implantasyon. Boncuk implantasyon araştırmacı inhibe veya ilgi alanında yerel genleri ikna etmek için yerel boncuk boncuk üzerine bir inhibitörü veya faktör ıslatın ve sonra implant sağlar. Bu genellikle Affigel veya heparin boncuklar (Şekil 2A) kullanılarak yapılır. Örneğin, kemik morfogenetik proteinleri (BMP) yerel kemik 4, 15 türetilmiş nöral krest indüksiyon sırasında önlenebilir veya gelişmekte olan ekstremitede 13 tomurcuk. Bir kordon implante edildikten sonra embriyo inkübatöre geri edilebilir ve geliştirmeye devam edecektir. Bu araştırmacılar, sklerotik halkada skleral kemik parçası oluşumunun engellenmesi (Şekil 2B ve 2C), 15 olarak, (bu durumda BMP), belirli bir genin engellenmesi alt baş etkilerini görmek için izin verir. Benzer şekilde, bariyer kolayca doku-doku sinyal ve boyalar mikroenjeksiyon de kolayca yapılabilir incelemek üzere ince kağıt tabakası arasına sokulabilir. </p&gt; <img alt="Şekil 1," src="/files/ftp_upload/52129/52129fig1.jpg" /> Embriyo ex ovo Görüntüleme Şekil 1.. (A) HH evre kabuk çıkarıldıktan sonra 20 civciv embriyo, vaskülarize embriyonik membranlar ile embriyo ok puan. (B) A HH evre açık nem odasında 40 embriyo. (C) HH evre erken ekstremite tomurcukları (ok) gösteren 35 embriyo. (D), göz (ok) olarak yapıların daha sonra sahne gelişme gösteren Sahne HH 41 embriyo. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52129/52129fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52129/52129fig2.jpg" /> Noggin Şekil 2. Örnek İmplantasyon Experim boncuk batırılmışent 4. (A) Embriyo HH evre 35 boncuk implantasyonu için altta yatan göz erişmek için örten zarlarda yırtık küçük bir delikten maruz. Bir konjonktiva papilla bitişik Affigel boncuk Yüksek büyütme içerlek gösterilir. C Ölçek çubuğu 75 mm. (B) alkalen fosfataz kemiklerinin halka boşluğu gösteren Noggin boncuk implantasyonu (ok), kemiklerinin halka hiçbir bozulma gösteren HH 38 (C) AP lekeli sol göz (kontrol sonrası sağ gözünü lekeli ), bu deney 38. Ayrıntılar Duench ve Franz-Odendaal 4 verilmiştir SS. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52129/52129fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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).

Optimize<em&gt; Ex-ovo</em&gt; Kalkınma Gelişmiş Aşamaları Chick Embriyolar ekimi

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

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Universidad Científica del Sur

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JoVE Journal

Developmental Biology

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

13.6K Görüntüleme.  Mount Saint Vincent University. 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). 

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

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Civciv Embriyo Özü murin Sinir Crest Kök Hücre Yetiştiriciliği Üretimi

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Nörobilim

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

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 morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

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

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Uzun vadede bir Batık Filtre Kağıdı Sandviç<em&gt; Ex Ovo</emErken Civciv Embriyolar&gt; Time-lapse Görüntüleme

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 ...

Gelişim Biyolojisi

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.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

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

Biyoloji

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Hızlı ve Nicel Intravital Chick Chorioallantoic Membran Modeli Kullanılarak Metastatik Regülatörlerin Keşfi

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Kanser Araştırmaları

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Yalıtım embriyonik doku ve timus örnek kullanarak bıldırcın-tavuk Chimeric organ oluşumu

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

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.

Civciv Ex ovo Kültür ve Ex ovo CAM Testi: Gerçekten Nasıl Çalışır?

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 ...

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Pregastrülasyondan İleri Organogenez'e Fare Embriyolarının Kültürü

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

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Tarafından Eski Tavuk Embriyolar içine Gen Transferi<em&gt; Ex ovo</em&gt; Elektroporasyon

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

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