Name: chick ex ovo culture and ex ovo cam assay: how it really works
Uploaded: 2009-11-30T14:45:00
Description: Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

Les auteurs tiennent à remercier J. et J. Kueper Wittschier d&#39;assistance technique excellente et le professeur Ruebben pour alimenter généreusement matériel tumoral.

Tout le matériel et les réactifs doivent être achetés stérile ou doit être stérilisé à la vapeur ou de la chaleur ou stérilisé avec de l&#39;EtOH 70%. Les auteurs indiquent que les expériences sur les animaux ont été effectuées en conformité avec les Communautés européennes Directive du Conseil (86/609/CEE), en suivant les directives du NIH concernant le soin et l&#39;utilisation des animaux à des procédures expérimentales et les règlements établis par l&#39;Animal ins...

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Res. 20, 237-239 (1992).</li><li>Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+generates+a+more+invasive+phenotype+of+tumour+cells:+an+in+vivo+experimental+setup+based+on+the+chorioallantoic+membrane.">Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane.</a> Pathol. Oncol. Res. , (2008).</li><li>Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. 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U., Krahn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+blood+levels+of+human+S100+in+melanoma+chick+embryo+xenografts'+circulation.">Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation.</a> Pigment Cell Res. 14, 9-13 (2001).</li><li>McFall, R. C., Sery, T. W., Makadon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+continuous+cell+line+derived+from+a+human+retinoblastoma.">Characterization of a new continuous cell line derived from a human retinoblastoma.</a> Cancer Research. 37, 1003-1010 (1977).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Research. 65, 10959-10969 (2005).</li><li>Kim, J., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li></ol>

Novateur ou juste un autre protocole de culture poussin? Avec les anciens protocoles de culture sans coquille 1-4 le taux de survie total des embryons de poulet ont été faibles, par exemple ca. 30% 1. Le protocole ex ovo culture raffinée décrit dans cet article la vidéo, en revanche, permet des taux de survie de plus de 50%. Par rapport à la musique classique dans des cultures in ovo culture ex ovo d&#39;embryons de poulet facil...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"><div><table border="1"><thead><tr><th> Nom du produit </th><th> Type </th><th> Entreprise </th><th> Numéro de catalogue </th><th> Commenter </th></tr></thead><tbody><tr><td> Les œufs fécondés </td><td> Animal </td><td> Soires Trockels, Allemagne </td><td></td><td></td><tr><td> Souris </td><td> Animal </td><td> Charles Rivers Laboratories </td><td></td><td></td><tr><td> Incubateur Type 3000 avec plateau à oeufs rotation </td><td> Outils </td><td> Siepmann GmbH Allemagne </td><td> 9503 </td><td> Maintenir à 37 ° C avec une humidité relative de 60% fixé au-dessus </td></tr><tr><td> Egg incubateur BSS 160 </td><td> Outils </td><td> Grumbach, Allemagne </td><td> 8101 </td><td> Maintenir à 37-387deg; C avec une humidité relative de 60% fixé au-dessus </td></tr><tr><td> PBS </td><td> Reagent </td><td> Sigma </td><td></td><td> PBS doit être froid (&gt; 4 ° C) et stérile </td></tr><tr><td> Eagle modifié par Dulbecco à moyen </td><td> Réactif </td><td> Sigma </td><td> D 6429 </td><td> Milieu de culture DMEM </td></tr><tr><td> Leibovitz L-15 Medium </td><td> Réactif </td><td> Invitrogen </td><td> 31415-029 </td><td> ici: moyen de collecte des échantillons de tumeurs </td></tr><tr><td> Tumeur spécifique à la cellule support </td><td> Réactif </td><td> Sigma </td><td></td><td> chaque groupe de recherche devrait utiliser le support qu&#39;ils culture de leurs cellules des tumeurs ou des milieux de culture communes, par exemple Leibovitz moyen ou milieu de Dulbecco modifié Eagle supplémenté avec 15% de sérum de veau fœtal (FBS), 4 mM de L-glutamine et 50 uM d&#39;insuline </td></tr><tr><td> EtOH à 70% </td><td> Réactif </td><td> Sigma </td><td></td><td></td><tr><td> Agitateur magnétique, triangled 80 mm </td><td> Outil </td><td> VWR </td><td> 442-0391 </td><td> Un agitateur magnétique triangled sert bien à se fissurer ee oeufs coquille. </td></tr><tr><td> Pinces DUMONT N ° 5 </td><td> Outil </td><td> Outils Fine Science </td><td> 11252-30 </td><td> biseautés tiges très fines (0,05 mm x mm pointe 0,02) </td></tr><tr><td> Pinces DUMONT N ° 7 </td><td> Outil </td><td> Outils Fine Science </td><td> 11271-30 </td><td> tiges courbées (0,07 mm x 0,10 mm pointe) </td></tr><tr><td> Ciseaux à ressort, droite, 8cm </td><td> Outil </td><td> Outils Fine Science </td><td> 15000-00 </td><td> fines, petites lames droites </td></tr><tr><td> Beaux Ciseaux Iris, droites </td><td> Outil </td><td> Outils Fine Science </td><td> 14094-11 </td><td> Utilisez-le pour couper notre CAM </td></tr><tr><td> Ciseaux standards, droits, pointus / mousses </td><td> Outil </td><td> Outils Fine Science </td><td> 14007-14 </td><td> Utilisez-le pour la décapitation ou la dislocation cervicale </td></tr><tr><td> microlitre seringue TLLX 1702, 25 pl </td><td> Outil </td><td> Hamilton </td><td> 80222 </td><td> Utiliser pour l&#39;injection dans le corps vitré </td></tr><tr><td> Hamilton 33-G aiguille (15 mm) </td><td> Outil </td><td> Outils Fine Science </td><td> 18073-15 </td><td> Utiliser pour l&#39;injection dans le corps vitré </td></tr><tr><td> Scalpels stériles </td><td> Outil </td><td></td><td></td><td> Utilisez pour disséquer des échantillons de tumeurs </td></tr><tr><td> Cuillère petit drain </td><td> Outil </td><td> Geuder, Allemagne </td><td> 15758 </td><td> Utilisez-le pour le transfert d&#39;embryons de poulet petits </td></tr><tr><td> Conseil d&#39;administration de cire avec des broches de fixation </td><td> Outil </td><td> Self made </td><td></td><td> Utilisez-le pour fixer des animaux pour la dissection </td></tr><tr><td> Boîtes de Pétri 60 x 15 mm </td><td> Outil </td><td> Greiner </td><td> 628102 </td><td></td><tr><td> Des boîtes de Pétri 92 x 16 mm </td><td> Outil </td><td> Sarstedt </td><td> 82.1472 </td><td></td><tr><td> Boîte de Pétri stérile 100 x 20 mm </td><td> Outil </td><td> Greiner </td><td> 664160 </td><td> très profonde, avec des entretoises pour la ventilation </td></tr><tr><td> Gobelet </td><td> Outil </td><td></td><td></td><td> 300-600 ml </td></tr><tr><td> FALCON tubes </td><td> Outil </td><td> FALCON</td><td></td><td> 15 ml et 50 ml </td></tr><tr><td> Tubes Eppendorf </td><td> Outil </td><td> Eppendorf </td><td></td><td> 1,5 ml et 2 ml </td></tr><tr><td> 1ml de pipettes </td><td> Outil </td><td></td><td></td><td> Utilisez-le pour couper des anneaux en plastique pour l&#39;application de substances / cellules </td></tr><tr><td> Cathéter veineux périphérique (PVL) </td><td> Outil </td><td> Viggo ® </td><td></td><td> Utilisez-le pour couper anneaux en silicone dans le tube pour l&#39;application de substances / cellules </td></tr><tr><td> Pipettes et pointes </td><td> Outil </td><td> Eppendorf / Abimed </td><td></td><td></td><tr><td> Autoclavé filtres pliés 595 1/2, 110 mm de diamètre </td><td> Outil </td><td> Schleicher &amp; Schuell </td><td> 311643 </td><td> Carrier, qui a causé au moins une irritation de la CAM; utilisé dans le document </td></tr><tr><td> PTFE Pledget, non résorbables, 6,3 x 152,4 x 1,6 mm </td><td> Outil </td><td> santec-médicale </td><td> REF 277, LOT 832/511-1 </td><td> Transporteur; resul causé de faux positifsts </td></tr><tr><td> Hxdroxyethylcellulose Tylose H 100000 </td><td> Réactif </td><td></td><td></td><td> Transporteur mélangé avec Kollidon 17 PF; provoqué des résultats faussement positifs </td></tr><tr><td> Kollidon 17 PF </td><td> Réactif </td><td> BASF </td><td> S30200 </td><td> mélangés avec Hydroxyéthylcellulose; causées résultats faussement positifs </td></tr><tr><td> Collagène Biomatrix TISSUDURA </td><td> Outil </td><td> Baxter Healthcare SA </td><td> REF B2246000999999 </td><td> Transporteurs; causées résultats faussement positifs </td></tr><tr><td> Cycle des lamelles de verre, 11 mm </td><td> Outil </td><td> Assistent Allemagne </td><td> 1001/0011 </td><td> Transporteur; entraîne des résultats faussement positifs </td></tr><tr><td> Bovine Collagen éponge </td><td> Outil </td><td> Wyeth </td><td></td><td> Transporteurs; causées résultats faussement positifs </td></tr><tr><td> Membrane collagène résorbable Resodont </td><td> Outil </td><td> Resorba Soins des plaies Allemagne </td><td> LOT 280303 </td><td> Transporteurs; causées résultats faussement positifs </td></tr><tr><td>Non-tissés écouvillons </td><td> Outil </td><td> Fink &amp; Walter GmbH </td><td> REF 328132 </td><td> Transporteurs; causées résultats faussement positifs </td></tr></tbody></table></div>

chick ex ovo culture and ex ovo cam assay: how it really works

Les œufs de poule dans la première phase de reproduction sont entre données in vitro et in vivo et de fournir un environnement de test vasculaire non seulement d&#39;étudier l&#39;angiogenèse mais aussi d&#39;étudier la tumorigenèse. Après que la membrane chorio-allantoïde de poulet (CAM) a mis au point, son réseau de vaisseaux sanguins peut être facilement accessible, manipulé et observé et permet donc un réglage optimal pour les essais d&#39;angiogenèse. Comme le système lymphoïde n&#39;est pas complètement développée jusqu&#39;aux stades tardifs de l&#39;incubation, l&#39;embryon de poulet sert d&#39;hôte immunodéprimé naturellement capable de soutenir les tissus et les cellules greffées sans restrictions spécifiques à l&#39;espèce. En plus de nourrir le développement d&#39;allo-et xénogreffes, le réseau de vaisseaux sanguins CAM offre un contexte favorable à la intravasation cellules tumorales, la diffusion et l&#39;arrestation vasculaire et un référentiel dans lequel les cellules arrêtées s&#39;extravaser pour former des micro foyers métastatiques. À des fins expérimentales, en moer des études récentes, le CAM a été exposée en coupant une fenêtre à travers la coquille d&#39;oeuf et expériences ont été réalisées in ovo, ce qui entraîne des limitations significatives dans l&#39;accessibilité de la CAM et possibilités pour l&#39;observation et la documentation photographique des effets. Quand le shell-moins des cultures de l&#39;embryon de poulet ont été utilisés 1-4, pas de détails expérimentaux ont été fournis et, si publiées, les taux de survie de ces cultures étaient faibles. Nous avons affiné la méthode de culture in ovo ex d&#39;embryons de poulet de façon significative par l&#39;introduction d&#39;une extrusion contrôlée rationnelle de la teneur en œuf. Ces cultures ex ovo améliorer l&#39;accessibilité de l&#39;embryon CAM et Chick, ce qui permet facilement dans la documentation in vivo des effets et de faciliter la manipulation expérimentale de l&#39;embryon. Cela permet à l&#39;application réussie d&#39;un grand nombre de questions scientifiques: (1) Comme un essai d&#39;angiogenèse amélioration de 5,6, (2) un dispositif expérimental mis en place pour les injections de faciliter l&#39;établissement ee vitré de l&#39;oeil d&#39;embryon de poulet 7-9, (3) comme un environnement de test pour la diffusion et la dispersion de intravasation cellules tumorales à partir de lignées cellulaires établies inoculées sur la CAM, 10-12 (4) en tant que système de maintien amélioré pour la transplantation réussie et culture de bourgeons de membres de poulet et 13 souris ainsi que (5) pour le greffage, de propagation, et de nouveau la greffe de tissu tumoral solide primaire obtenu à partir de biopsies de la surface de la came 14. Dans cet article, nous décrivons la vidéo mise en place d&#39;une culture raffinée poussin ex ovo et le dosage CAM avec des taux de survie de plus de 50%. En plus nous offrons une démonstration étape par étape de l&#39;application réussie de la culture ex ovo pour un grand nombre d&#39;applications scientifiques. Daniel S. Dohle, Susanne D. Pasa, et Sebastian Gustmann contribué également à cette étude.

Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

La membrane chorio-allantoïde de poulet (CAM) est un environnement unique et naturellement immunodéficients culture favorable pour étudier l&#39;angiogenèse et de la tumorigenèse. Cet article vidéo montre les différentes étapes de poussin<em&gt; Ovo ex</em&gt; Culture, à l&#39;application de substances potentiellement angiogéniques et l&#39;inoculation des cellules tumorales avec succès et les tissus sur la surface de la came.

Poussin<em&gt; Ovo ex</em&gt; Culture et<em&gt; Ovo ex</em&gt; CAM Test: Comment il fonctionne vraiment

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 ...

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Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

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.

En électroporations Ovo d&#39;embryons au stade de poulet 10 HH

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 ...

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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.

Méthode pour la culture d&#39;embryons de poulet Early ex vivo (Nouvelle 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 ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Décrypter Pathways axonale des groupes génétiquement définis de neurones dans le tube neural Chick Utilisant In ovo L&#39;électroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

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.

Une Ex-ovo- Poulet embryonnaire du système de culture approprié pour les applications d&#39;imagerie et de microchirurgie

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

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

En électroporation Ovo dans la rétine de poussin embryonnaire

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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.

Transfert de gènes dans des embryons de poulet âgés par<em&gt; Ex ovo</em&gt; Électroporation

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Méthode de la culture pour le embryonnaire Souris Coeur

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Culture de la peau de souris embryonnaires et Live-imagerie de la migration mélanoblaste

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.