Vorremmo ringraziare Paul Poirier, il produttore di media, a Mount Saint Vincent Università per il suo lavoro in ripresa e modifica la parte video di questo manoscritto. Riconosciamo Scienze Naturali e di Ingegneria Research Council del Canada per il finanziamento.

Nota: Tutte le forniture sono elencati nella tabella 1. 1. Memorizzare il pollo embrioni <ol><li> Incubare uova di gallina del ceppo Gallus gallus orizzontalmente a 37 ° C con circa il 40% di umidità e girare le uova una o due volte al giorno. Passando uova è importante per evitare l&#39;embrione di aderire al guscio d&#39;uovo. </li><li> Non per non trasformare l&#39;uovo in 24 ore prima di impostare la cultura, altrimenti l&#39;embrione sarà situato ventrale alla massa tuorlo e verrà danneggiato aprendo l&#39;uovo al punto 3. Inoltre, tenere le uova a 4 o C ° per non più di una settimana prima di incubazione di sviluppo &quot;halt&quot;, ma questo non è l&#39;ideale. </li></ol> 2. Staging pollo Embrioni <ol><li> Fase gli embrioni di pollo con l&#39;Hamburger e Hamilton 12 tabella di gestione temporanea. Il palcoscenico ideale per l&#39;impostazione della coltura ex ovo è HH fase 19-20 (circa 3 giorni di incubazione) becausa questo è poco dopo la testa gira a 53 HPF. Nota: HH fase 19-20 è caratterizzata dalle seguenti caratteristiche morfologiche: somiti sono estese nella maggior parte della coda, ma alla fine della coda rimane non segmentato, la gemma coda è arricciata, allantoide è piccolo e ha limitato vascolare, le gambe gemme sono più grandi delle ali gemme e gli occhi sono pigmentato o hanno una tonalità grigiastra. </li></ol> 3. Rimuovere l&#39;embrione da Shell <ol><li> Prima di rimuovere l&#39;embrione dal guscio, spruzzare il guscio con il 70% di etanolo e lasciare asciugare. Non girare l&#39;uovo rapidamente per non danneggiare l&#39;embrione. </li><li> Facendo attenzione a non alterare l&#39;orientamento dell&#39;uovo, crepa accuratamente l&#39;uovo sul lato inferiore (cioè, il lato che era ventrale durante l&#39;incubazione) e rilasciare l&#39;embrione in un sterile pesano barca (88 x 88 x 23 mm). Pulire pesare barche con il 70% di etanolo. Controllare l&#39;embrione di garantire che il sacco vitellino non sia danneggiato. Se il tuorlo hcome rotto, scartare l&#39;embrione, in quanto non sopravviverà. </li><li> Osservare l&#39;embrione per assicurarsi che sia praticabile; il cuore batte, vasi appare normale, e evidenti anomalie sono presenti. Assicurarsi che i bordi della barca pesare siano asciutte. </li><li> Utilizzando una pipetta, goccia 40 ml di penicillina / streptomicina (5.000 unità penicillina, 5 mg di streptomicina per ml) sulla parte superiore del dell&#39;albumina per aiutare a prevenire l&#39;infezione. </li></ol> 4. Preparare la Camera Umidità <ol><li> Inserire una piccola pila di Kim-salviette e / o un tampone assorbente in cotone sul fondo di un contenitore di plastica sterile (12 x 12 x 6 cm) strofinando con etanolo al 70%. </li><li> Aggiungi sterili, acqua distillata per inumidire i Kim-salviette e / o cotone (circa 150 ml di acqua). </li></ol> 5. Assemblaggio della Ex Ovo Cultura <ol><li> Posizionare la barca pesare contenente l&#39;embrione sopra dell&#39;imbottitura umida. Quindi, posto a metà di una sterile petri-piatto quadrato (9.5 x 9.5 cm) sulla parte superiore del tsi pesano barca per formare un coperchio allentato. </li><li> Coprire il contenitore di plastica con il suo coperchio. Premere giù due angoli del coperchio, assicurando una tenuta parziale che consente ancora una buona circolazione dell&#39;aria all&#39;interno della camera. </li><li> Posizionare con cura l&#39;installazione in 37 o C incubatore fino stadio desiderato. </li><li> Posizionare i contenitori di acqua in incubatrice per aiutare a controllare l&#39;umidità, se un incubatore umidità controllata non è disponibile. Sterilizzare tutta l&#39;acqua nelle camere di umidità e nell&#39;incubatrice e riempire come necessario durante il periodo di incubazione. 40% umidità è ideale. </li></ol>

<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>

Gli autori non hanno interessi finanziari concorrenti per quanto riguarda le informazioni presentate in questo manoscritto.

Ex ovo coltura e di finestre entrambi hanno vantaggi e le sfide. Qui mettiamo a confronto i vantaggi e le sfide del bicchiere di polistirolo ex ovo metodo e il metodo a finestre al nostro ex ottimizzato ovo metodo illustrato qui. Il nostro metodo permette la manipolazione e semplice osservazione del embrione di pollo in fase avanzata di sviluppo e dei nostri filtri alla ex ovo tradizionale metodo 1, 2, 3 rendono inoltre molto facile da usare in classi di laurea lab...

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.

Questo ex ovo metodo consente l&#39;osservazione di embrioni dalle prime fasi di sviluppo (HH 19/20) in fase avanzata di sviluppo (HH 40-41) (Figura 1A e 1B). Impostazione della cultura a HH 19-20 aumenta sopravvivenza degli embrioni nella cultura. Prima della testa di svolta (prima 53 HPF) di sopravvivenza è molto bassa nella cultura e dopo la tappa 21, l&#39;embrione tende ad attaccare di più per la shell alla rimozione così si ottengono un numero inferiore di embrioni int...

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).

Ottimizzato<em&gt; Ex-ovo</em&gt; Coltura di Chick embrioni a stadi avanzati di sviluppo

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

optimized-ex-ovo-culturing-chick-embryos-to-advanced-stages

Research

JoVE Journal

Developmental Biology

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

13.5K Views.  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.

Produzione di estratto di Chick dell&#39;embrione per la coltivazione delle cellule della cresta neurale Murine staminali

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

Ricerca

Neuroscienze

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.

Un sommerso Sandwich carta da filtro per il lungo termine<em&gt; Ex Ovo</em&gt; Time-lapse imaging di Chick primi embrioni

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 ...

Biologia dello sviluppo

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.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

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

Biologia

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.

Scoperta di regolatori metastatici utilizzando un modello di membrana corioallantoica di pulcino intravitale rapido e quantitativo

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

Ricerca sul cancro

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.

Metodo di coltura organotypic per studiare lo sviluppo dei tessuti embrionali di pollo

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

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.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

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.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

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 Coltura di embrioni di topo dalla pregastrulazione all'organogenesi avanzata

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

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

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 the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

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

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

Metodi Ovo ed Ex Ovo per studiare lo sviluppo dell'orecchio interno aviario

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 ...

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.

Il trasferimento genico in embrioni di pollo da anziani<em&gt; Ex ovo</em&gt; Elettroporazione

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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