This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

Etik Beyanı: Bütün bu deneyler hayvan bakımı ve Nottingham Üniversitesi etik kurallara uyun. Tırnak için Heparin Boncuk 1. Hazırlık <ol><li> Kullanmadan önce PBS içinde iyice heparin boncuk yıkayın. Not: boncuk PBS içinde bir çamur olarak 4 ° C &#39;de muhafaza edilebilir. <ol><li> PBS, 1 ml damla bir 20 ul pipet ile stoktan bunları kaldırarak aşılama için boncuk seçin. Daha sonra 3 santimetrelik bir petri tabağına PBS 20 ul damla içine boncuk aktarmak için 2 ul ayarlanmış bir mikropipet kullanın. P2 pipet ile seçilen boncuklar transfer diseksiyon mikroskobu stereo kullanarak, boyutuna göre boncuk seçin ve; Bu yaklaşık 100 mikron çapında idealdir. </li></ol></li><li> Boncuk PBS çıkarın. 20 ul pipet ve ince bir saatçi pensesi ile kılcal hareket ile artık sıvı sıvının en çıkarın. Not: fiili büyüme yüzden mümkün olduğunca ortadan kaldırmak için önemlidirbu boncuklar ilave edildiğinde r seyreltilmez. </li><li> Boncuklar üzerine, doğrudan büyüme faktörü Pipet. FGF18 için PBS içinde% 0.1 BSA içinde 0.5 mg / ml&#39;de yeniden rekombinant FGF18 0.5 ul ekle. Buharlaşan sıvının önlemek için çanağın kenarları etrafında birkaç damla su koyun. </li><li> Oda sıcaklığında 1 saat için boncuklan inkübe edin. Pasteur pipeti ile çanak su damlalarını ve aşılama için hazır buz üzerine yerleştirin. </li><li> Onlara görselleştirmek yardımcı olmak için aşılama öncesi (saydam boncuk için) aktarım işlemlerinin% 2 fenol kırmızısı 4-5 boncuk Gerekirse. Embriyo transfer olmadan önce PBS içinde durulayın. </li></ol> Tırnak için AG 1-X2 Boncuk 2. Hazırlık <ol><li> 0,2 N formik asit ile Türevlendirilen boncuk Kullanımdan önce. <ol><li> Bu kullanım, 1.5 ml mikrosantrifüj tüp boncuk aktarmak için bir spatula yapmak için. 0,2 N formik asit 1 ml ilave edin ve 1 saat süre ile bir çalkalama platformu üzerinde yıkayın. </li><li> Sonra P1000 pipet ile formik asit çıkarın ve 1 ile değiştirinml su. 1 saat yıkayın ve formik asit kalan çıkarmak için altı kez tekrarlayın. </li><li> P1000 pipet ile kalan sıvıyı çıkarın ve 4 ° C&#39;de boncuk saklayın. Not: Bu aşamada bütün artık suyun ayrılması için gerekli değildir. </li></ol></li><li> 3 cm&#39;lik bir petri kabına küçük bir spatula kullanılarak stoktan yaklaşık 5-10 ul hacmi tanelerin küçük pelet çıkarın ve DMSO&#39;nun 1 ml ekleyin (ya da ilaç içinde çözünmekte çözücü hangisi geçerli ise). Not: Bu doğru boyutta olanlar seçmek için hangi birkaç yüz boncuk sağlamalıdır. </li><li> Sonra başka bir 3 santimetrelik bir petri tabağına bir çözücü 20 ul damla yaklaşık 100 um çapında boncuklar aktarmak için 2 ul ayarlanmış bir P2 pipet kullanın. </li><li> 20 ul pipet ve 2 ul pipet ile herhangi bir kalıntı çözücü ile çözücü çıkarın. İlacın 20 ul uygulanacak değiştirin. Not: tutarlılığı (ya da hiç çözücü kullanılır) DMSO içinde 100 uM bir konsantrasyonda ilaç çözünmesi için,Bazı küçük molekül inhibitörleri olarak -80 ° C de 20 ul ve deposuna kısım kararsızdır ve tekrarlanan donma-çözülme önlemek için en iyisidir. Gerekirse o boncuk iliklerine önce istenilen konsantrasyonda ilacın sulandırmak. Etkili konsantrasyon, her bir ilaç için ampirik olarak tespit edilmesi gerekebilir. </li><li> 1 saat süreyle inkübe edin. Bu moleküllerin çoğu ışığa duyarlı olarak ışıktan koruyun. </li><li> Suda çözünmüş% 2 fenol kırmızısı 20 ul içine 2 ul ayarlanmış bir p2 pipet ile aktarım işlemlerinin 4-5 boncuk aşılama önce. Boncuk görselleştirmek için bir stereo mikroskop kullanarak bu adımı gerçekleştirin. İnce saatçiler forseps ile tek bir boncuk veya bir tel döngü çıkarın ve embriyo aktarmadan önce PBS içinde yıkayın. Bu faaliyetin kaybına neden olabilir kullanımdan önce fazla 15 dakika 2% fenol kırmızısı boncuk bırakmayın. </li></ol> 3. Tungsten İğneleri hazırlanması <ol><li> 3-4 cm uzunlukta ince tungsten tel kesin. Ucuna tel tek parça takınBunsen beki bir cam Pasteur pipeti ve eritebilir pozisyonda sabitlemek için. Bunlar, kullanım sırasında zarar görmüş, yedek olmadığından emin olmak için 10 iğne kadar hazırlayın. </li><li> Bir blowtorch ateşe telin ucunu yerleştirin ve (2-3 dk civarında) tungsten beyaz parlıyor ve ucu keskin kadar orada tutun. Not: Kullanım sırasında iğne temizlenebilir ve gerektiğinde blowtorch yeniden bilenmiş. NOT: Bu iğneler boncuklar aktarmak için döngüler yapmak için kullanılabilir. Yavaşça tezgah iğne dokunmak ve bir döngü oluşturacak şekilde viraj olacak. </li></ol> Boncuk Greft 4. hazırlanması Embriyolar <ol><li> Istenen sahne (en uzuv tomurcuk manipülasyonları için 3-5 gün) ulaşıncaya kadar kör uç yukarı gelecek şekilde yumurta kuluçkaya yatmaktadır. Kabuğunu kırmak ve daha sonra kabuk kaldırmak ve embriyo maruz forseps kullanmak künt ucunda künt forseps musluk kullanma. Yumurta kabuğu zarı emin olun da kaldırılır. Not: PBS 1-2 ml yardımcı olmak için bir Pasteur pipeti ile eklenebilirBu zar sarısı yapışıyor eğer. Yavaşça, sarısı yüzeyine PBS pipetle sarısı zarar vermemeye özen forseps ile membran tutup, yavaşça yumurta çekin. </li><li> 10 ml şırınga ile yumurta ve 19 G iğne albümin 5 ml kadar kaldırın. Embriyo sağkalım oranları azaltabilir büyük hacimlerin çıkarılması gibi yumurta mühürlemek için kullanılan bant ile temas yapmaz embriyo sağlamak için gerekli olan tek kadar götür. </li><li> Embriyo 15 100 U penisilin içeren PBS ml ve 0.1 mg streptomisin / ml sanatçılar india mürekkep 5-6 damla görselleştirmek için. 0.5-1 enjekte ml doğrudan 1 ml şırınga ile embriyo ve 25 G iğne altında. </li><li> Dihidrat etmez embriyo sağlamak için yumurtanın içine% 1 fetal buzağı serumu ve 100 U penisilin ve 0.1 mg streptomisin / ml PBS 2-3 ml ekleyin. Bilenmiş saatçiler forseps kullanarak vitellin membran kaldırmak ve gelişmekte olan ekstremite tomurcukları üzerinde amniyon açın. Embriyo kurumaz emin olun veGerekirse, daha fazla PBS / FCS ekleyin. NOT: bacak tomurcukları açıkça embriyonun kanadında eşleştirilmiş çıkıntılar olarak görülebilir. Bu aşamalarda embriyo böyle sağ tarafı normal üst olduğunu dönecek. Sonuç olarak, doğru ayağının içine doğru geriye boncuk greft ve karşı, kontrol olarak bırakıldı olanları kullanmak için, normalde daha kolaydır. </li><li> Boncuk implante edilecek uzuv tomurcuk içine bir kesi yapmak için bir bilenmiş tungsten tel kullanın. Genç aşamalarında bu zor olmasına rağmen mümkün ekstremite tomurcuk aracılığıyla tüm yol kesme kaçının. NOT: Büyük embriyoların (HH evre 22 ve üzeri) yılında aşılama için ama uzuv kendisi küçük tomurcuk olarak bu daha zor genç aşamalarında bacak tomurcuk belirli bölgeleri seçmek mümkündür. O bacak tomurcuk, yani özellikle genç aşamalarında, boncuk proksimal ekstremite kalır aşılı boncuk geçmiş büyüyecek hatırlamak da önemlidir. </li><li> Ya ekstra ince wa kullanarak büyüme faktörü veya ilaç ya bir boncuk Pick uptchmakers forseps veya tungsten iğne yapılmış bir döngü. Boncuk DMSO veya fenol kırmızısı ya batırılmış edilmişse, embriyo başvurmadan önce PBS içinde yıkayın. Forseps / tel döngü ile embriyo boncuğu aktarın. Sonra kesi içine boncuk eklemek için bilenmiş tungsten tel kullanın. </li><li> Embriyo hidrate tutulmasını sağlamak yumurta PBS / FCS 1-2 ml ekleyin, ancak bu embriyolar zarar ve boncuk yerinden neden olabilir olarak embriyo kendisi doğrudan uygulamaktan kaçının. Bantla yumurta Seal ve inkübatör dönmek. </li></ol>

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The authors have nothing to disclose.

Ovo dokuların geliştirilmesi için doğrudan uygulanabilir boncuk greft kullanımı, uygulandıkları hangi gelişim aşamasında ve pozlama süresi boyunca eşsiz kontrol vererek gelişim sırasında büyüme faktörü sinyalizasyon rolünü incelemek için güçlü bir araçtır. Molekülünün her türü için boncuk seçimi önemlidir. Her ampirik bu boncuklar üzerinde inhibitör etkinliğini test etmek için gerekli olmasına rağmen, burada anlatılan önleyicileri ve retinoik asit ...

Kuş embriyolar yıllardır 1 gelişim çalışma için güçlü bir araç sağladı. Bunların en yararlı özelliklerinden biri bunların manipüle edilmeleri nispeten kolay olmasıdır. Dış gelişimi, hücre kaderine 2,3 çalışmak için klasik bir bıldırcın piliç kimera sistemi olarak örnekler dahil olmak üzere çeşitli micromanipulations embriyo ulaşmak için yumurta açıp gerçekleştirmek mümkün kılar, geliştirme 4,5 sırasında belirli dokularda aşırı ekspresyona yönelik retrovirüslerin enjeksiyonu ve eksplant kültürü gelişimsel sinyal kaynaklarını 6 tanımlamak için. Daha yakın zamanlarda GFP ifade transgenik tavuk hattından greft ile etiketsiz bilgisayarlar arasında oluşturulan kimeralardır klasik aşılama ve genetik modifikasyon kombinasyonu gelişimi 7,8 dair önemli bilgiler sağlayabilir göstermiştir. Kuş embriyo manipüle edilebilir kolaylığı uzuv incelemek için o mükemmel bir model yapmıştırgelişimi 9. In vivo gelişmekte bacaklarda özgü büyüme faktörlerinin uygulanması bacak desenlendirme 10,11 değiştirebilir ve bu sürecin 12 içgörü sağlamaya devam faktörlerin belirlenmesinde etkili olmuştur. Bu yaklaşım aynı zamanda, kas gelişimini düzenleyen faktörleri incelemek için kullanılır olmuştur ve böyle Wnts 13, BMP 14 ve HGF 15 gibi sayısız sinyaller için roller ortaya çıkardı. Son zamanlarda bu teknik, bacak tomurcuk miyojenik gen ekspresyonunu kontrol sinyalleri araştırmak için kullanılmıştır ve FGF18 ve retinoik asit arasındaki etkileşimler MyoD ekspresyonu 16 zamanlamasını kontrol göstermiştir. Boncuklar üzerine yüklendi ve daha sonra belirlenen gelişim aşamalarında spesifik dokular doğrudan aşılanabilir büyüme faktörleri ve küçük moleküllerin bir kombinasyon kullanılarak gelişimi esnasında hemen hemen her zaman ve bölge müdahale imkanı verir. Bu inve için kullanılmıştırhücre gruplarının desenlendirme 17,18, nöral şartnamede 19, nöral krest göç 20 ve eksen uzantısı 21 dahil olmak üzere birçok süreçleri stigate. Burada tavuk bacaklarda gelişmekte olan ya da büyüme faktörleri ya da inhibitörlerin batırılmış boncuklar aşılama için bir yöntem tarif eder. Bu in situ hibridizasyon ile, kas spesifik gen ekspresyonunu analiz ederek Miyogenesis bu sinyallerin etkisini belirlemek amacıyla kullanılmıştır. Bu tür retinoik asit veya belirli bir sinyal yollarının küçük molekül inhibitörleri olarak küçük hidrofobik moleküller için büyüme faktörleri ya da AG 1-X2 boncuklar için kullanılan heparin batırılmış boncuklar, kullanılarak greft tarif eder. Bununla birlikte, diğer boncuklar da FGF&#39;ler 22 ve 23, her iki Shh teslim etmek için kullanılmıştır temin edilebilmektedir.

<table><tbody><tr><td>Heparin acrylic beads</td><td>Sigma</td><td>H5263</td><td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td></tr><tr><td>AG 1-X2 beads</td><td>Bio-Rad</td><td>140-1231</td><td></td></tr><tr><td>Affi Gel blue beads</td><td>Bio-Rad</td><td>153-7301; 153-7302</td><td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td></tr><tr><td>FGF18</td><td>Peprotech</td><td>100-28</td><td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&amp;deg;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td></tr><tr><td>U0126</td><td>Cell Signalling</td><td>9903</td><td>Make to 20mM stock in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>BMS-493</td><td>Tocris Biosciences</td><td>3509</td><td>Resuspend in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>Black Indian ink</td><td>Windsor and Newton</td><td>5012572003384 (30 ml)</td><td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td></tr><tr><td>Tungsten wire, 0.1 mm dia. 99.95%</td><td>Alfa Aesar</td><td>10404</td><td></td></tr><tr><td>Penicillin / streptomycin</td><td>Sigma</td><td>P0781</td><td>Dilute 100X in PBS/ink and PBS/FCS</td></tr></tbody></table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

Tavuk embriyo kullanarak doğrudan büyüme faktörleri ya da gen ekspresyonu ve sinyal transdüksiyon yollarının aktivasyonu ile ilgili sinyal yollarının spesifik inhibitörleri ya da etkilerini test etmek mümkündür. Bu teknik, belirli zamanlarda için kesin olarak tanımlanmış gelişim aşamalarında sinyal molekülleri teslim izin verir. Bu embriyolar, hasat edilmiştir ve gen ekspresyonu in situ melezleme ya da immün ile gözlemlenen sinyal transdüksiyon yollarının aktivasyonu, örneğin, incelenebilir sonra. U0126, bir MEK inhibitörü batırılmış bu FGF18 batırılmış bir video heparin boncuk veya AG 1-X2 boncuk, ovo ekstremite tomurcuk içine aşılı edilir. Bu FGF18 U0126 tarafından inhibe edilir MyoD ve ERK fosforilasyonu ve endojen ve FGF18 kaynaklı hem MyoD ekspresyonunu uyardığını göstermektedir. Bir retinoik asit antagonisti batırılmış Boncuk FGF18 tarafından prematüre MyoD indüksiyon potansiyalize edebilir. Bu yaklaşım bize olabilirFarklı büyüme faktörleri ve inhibitörleri ve geniş bir yelpazesi ile ed de gelişmekte olan embriyonun diğer dokularda uyarlanmıştır.

HH aşamada 21 MyoD boyama gelişmekte Somitlerin (Şekil 1A) miyotom gözüyle görülebilir, ancak bacak miyoblastlar geliştirilmesinde ifade değildir. Şekil 1B, bir FGF18 kordon greft, aşağıdaki MyoD 6 saat boyunca in situ hibridizasyon gösterir. Karşı bacak bir ifade yoktur ise MyoD boncuk yakın miyoblastların indüklenir. U0126 batırılmış bir boncuk, MEK, MyoD (Şekil 1C) bloklar FGF18 indüksiyon spesifik bir inhibitörü olan Co-aşılama....

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Gen İfade Etkileyen Sinyal İletimi Pathways tanımak Tavuk Embriyo uzuvlar Geliştirme içine Boncuk eklenme

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

grafting-beads-into-developing-chicken-embryo-limbs-to-identify

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.8K Görüntüleme.  University of Nottingham. By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Video: Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

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.

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 ...

Gelişim Biyolojisi

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

ChickGFP Nöral Tüp Greftleme ve Karbosiyanine Boya DiI Enjeksiyonu Kullanılarak Tavuk Embriyoları İçinde Nöral Kret Hücrelerinin ve Kan Damarlarının Çift Etiketlenimi

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebra balığı Embriyo microbead İmplantasyonu

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

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.

Erken Evre Tavuk Embriyolar Büyüme Faktörü-Kaplı Boncuk yerleştirme

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

Biyoloji

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.

HH Sahne 10 Tavuk Embriyolar Ovo 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 ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

Aday Faktörü Soaked Boncuk ile birlikte Geçirimsiz Engelleri Uygulama Chick Endüktif Sinyalleri Eğitim için

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Assay for Neural Induction in the Chick Embryo

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

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

Civciv Embriyo Sinir İndüksiyon için Testi

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

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 ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

Morfogenez, Desenleme ve Hücre Farklılaşmasında Erken Adımları Anlamak için Tavuk Rekombinant Uzuvları Testi

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

Islatılmış Boncuk Tahlili Kullanılarak Tavuk Embriyogenezi Sırasında Hücre Farklılaşması, Morfogenez ve Desenleme Analizi

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Gen İfade Etkileyen Sinyal İletimi Pathways tanımak Tavuk Embriyo uzuvlar Geliştirme içine Boncuk eklenme

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Chick^GFPNöral Tüp Greftleme ve Karbosiyanine Boya DiI Enjeksiyonu Kullanılarak Tavuk Embriyoları İçinde Nöral Kret Hücrelerinin ve Kan Damarlarının Çift Etiketlenimi