Nous tenons à remercier M. Enver Maaz pour l&#39;utilisation de son appareil photo HD (Cannon VIXIA HFS100). Ce travail a été soutenu en partie par des subventions des NIH (EY018738), New Jersey Commission sur la recherche sur la moelle épinière (08-3074-RCS-E-0 et 10-3091-RCS-E-0), et le Prix Busch recherche biomédicale.

Depuis quelques parties du protocole sont réalisées comme décrit précédemment dans d&#39;autres vidéos et des documents de Jove 2 3 avec des modifications mineures, nous ne discuterons pas ceux en détail ici. 1. La manipulation des œufs et de Préparation à aiguilles <ol><li> Les œufs peuvent être stockés dans un refroidisseur de vin à environ 13 ° C pendant 1 semaine. Si la température est trop élevée, les embryons commence à se développer anormalement, tandis que la baisse de température provoque une mortalité élevée. Une fois que vous êtes prêt à prendre incuber les oeufs à partir du refroidisseur de vin et de mettre les œufs à la verticale avec la plus grande extrémité vers le haut. Gardez les oeufs dans la température ambiante pendant au moins 2 heures avant de les mettre dans l&#39;incubateur à 37,5 ° C. </li><li> Incuber les œufs destinés à 96-100 heures, ce qui est d&#39;environ 4 à jour embryonnaire d&#39;obtenir des embryons qui sont à la Hamburger et le stade de Hamilton 22 4-5. </li><li> Préparer aiguilles micropipette à partir de tubes de verre tirés capillaires et de briser la pointe sous une dissection microportée avec une pince à épiler pour obtenir une ouverture d&#39;extrémité d&#39;environ 0,1 m de diamètre et d&#39;un cône de 20 mm. Aiguilles avec de grandes pointes ont de la difficulté à percer la membrane vitelline tandis que les petits conseils ont de la difficulté de chargement et de fournir la solution d&#39;ADN. </li><li> Fixer l&#39;aiguille à une seringue 0,1 ml Hamilton étanche au gaz monté sur un micromanipulateur. Utilisez un petit morceau de tube en silicone Masterplex pour fixer l&#39;aiguille à la seringue. Étant donné que les connexions sont bien serrées pas besoin d&#39;ajouter de l&#39;huile minérale pour sceller cet attachement. </li><li> Mélanger 2 pi de solution d&#39;ADN plasmide rapporteur avec une concentration allant de 3 à 6 pg / pl et 0,2 pi de vert rapide (0,025%) sur un morceau de parafilm. Rapide colorant vert aidera à visualiser l&#39;injection. Lentement, charger l&#39;aiguille avec le mélange. </li><li> Pour libérer la membrane vitelline de la membrane interne tourner l&#39;œuf délicatement environ 180 ° et d&#39;attendre pendant quelques minutes, puis le faire pivoter à sa position initiale et le mettre pour l&#39;électroporation. </li><li> Essuyez le forçageeps et coquille d&#39;œuf avec de l&#39;éthanol à 70% pour éviter l&#39;infection de l&#39;embryon. </li><li> Faites un petit trou sur l&#39;œuf immédiatement au-dessus de la cellule d&#39;air avec une paire de pince AA. Veillez à ne pas casser la coquille de l&#39;oeuf. Retirer les morceaux de petits un à la fois de faire une petite fenêtre. Retirer délicatement la membrane interne en utilisant les forceps, sans toucher la membrane vitelline. </li></ol> 2. Injection et l&#39;électroporation <ol><li> Pour éviter d&#39;endommager le cerveau ou le cœur, la position de l&#39;aiguille contre latéral du faisceau principal des vaisseaux sanguins pénètrent dans l&#39;œil et pointant vers le bec. </li><li> Pierce à travers la membrane vitelline, la sclérotique, la rétine et l&#39;humeur vitrée par un doux poussée soudaine de l&#39;aiguille. Si l&#39;aiguille perce l&#39;autre extrémité de l&#39;œil, il devrait bien se passer si elle endommage une veine de sang importante. </li><li> Tirez doucement l&#39;aiguille sur le bord de l&#39;ouverture et placez-le près de la tangente à la paroi extérieure du globe oculaire. </li><li> Insert l&#39;aiguille dans l&#39;espace sous la rétine entre la sclérotique et la rétine. </li><li> Injecter de l&#39;ADN jusqu&#39;à ce que vous pouvez visualiser la solution verte de remplissage du côté du globe oculaire et en poussant vers l&#39;intérieur la rétine par la création d&#39;un renflement. </li><li> Si votre mise aiguille n&#39;est pas correcte, alors vous verrez la solution d&#39;ADN propager à l&#39;intérieur du corps vitré en remplissant le milieu de la balle des yeux. Aussi, si vous endommager la rétine trop, vous verrez la solution d&#39;ADN est venue hors du globe oculaire. </li><li> Lentement retirer l&#39;aiguille et placer immédiatement les électrodes en parallèle à l&#39;intérieur de l&#39;œuf après trempage dans du PBS. Poussez vers le bas les électrodes à plonger dans le liquide amniotique d&#39;une manière telle sorte que l&#39;œil injecté est situé entre les électrodes. Évitez de toucher un vaisseau sanguin important ou le cœur avec les électrodes en les plaçant. L&#39;électrode négative devrait levera à le côté d&#39;injection de telle sorte que l&#39;ADN peut être transporté depuis l&#39;espace rétinienne sous-programme dans la rétine en direction d&#39;électrode positive. Electroporate la rétine avec 5 impulsions de 15V pour 50 ms avec des intervalles de 950 ms. </li><li> Retirez délicatement les électrodes et sceller la fenêtre de l&#39;œuf avec des morceaux de scotch clair. </li><li> Étiquetez et datez l&#39;œuf injecté avant de le remettre à l&#39;incubateur. En règle générale, il faut environ 3 à 5 minutes pour compléter le processus d&#39;électroporation ensemble. </li><li> Expression de la GFP peut être vu dès 8 heures après l&#39;électroporation. Cependant, vous pouvez attendre jusqu&#39;à ce que l&#39;embryon atteint le stade de croissance désiré avant la récolte. </li></ol> 3. Les résultats représentatifs Dans notre étude, nous utilisons diverses constructions plasmidiques pour étudier la régulation de l&#39;expression génique qui a impliqué le développement des cellules de la rétine. Dans cette vidéo pCAG-GFP (contrôle de transfection) a été utilisée pour suivre une injection réussie et l&#39;électroporation. Toutefois, toute construction plasmidique avec gène rapporteur (la GFP, DP etc) peut être utilisé. Même si expression de la GFP peut être vu dès 8 heures après élémentsctroporation, nous avons généralement commencer à récolter les oeufs le jour 6 (E6) et au-delà. Électroporé rétines ont été disséqués de l&#39;embryon et analysées sous microscope à dissection fluorescente avant inclusion et la coupe. En règle générale, l&#39;expression du gène rapporteur peut être vu au moins un quart de la rétine après une électroporation efficace (figure 1). Les tissus rétiniens transfectées ont été analysés par le biais de sectionnement pour la visualisation claire des morphologies cellulaires. L&#39;immunohistochimie utilisant des marqueurs de type cellule spécifique (Brn3a, Pax6 etc) a permis de caractériser l&#39;expression spécifique des cellules GFP (figure 2). <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> Figure 1. Électroporation réussie de journaliste résultats expression de la GFP plasmide positif. Poulet embryonnaire rétines ont été injectés et une électroporation à jour embryonnaire 4 et récoltés à jour embryonnaire 6. Au moins 25% de la rétine a été avec succès transfectées (A view = Haut, BVue de dessous =). Échelle Bar = 1mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> Figure 2. Caractérisation de la GFP exprimer cellules de la rétine à l&#39;aide immunohistochimie. GFP exprimant les tissus rétiniens au stade E7 étaient fixes et sectionné. Ces sections ont ensuite été colorées avec des marqueurs cellulaires spécifiques. Brn3a a été utilisée pour déterminer cellules ganglionnaires (Un) tout en Pax6 a été utilisée pour déterminer horizontale, amacrines et les cellules ganglionnaires (chambres d&#39;). Échelle = 50 microns.

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</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.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

Toutes les expériences sur les animaux ont été approuvés par le soin des animaux et du Comité institutionnel des installations à l&#39;Université Rutgers.

Ciblée de la rétine et d&#39;injection in ovo dans électroporation dans la rétine embryonnaire au stade E4 peut cibler spécifiquement les cellules progénitrices la rétine dues à la capacité de visualiser tous les six principaux types de cellules rétiniennes au niveau de la cellule unique. En électroporation in ovo à HH10 (~ E1.5) ciblant le vésicule optique est capable de transfecter des cellules qui se développent pour former l&#39;œil. Cependant, ces cellules ont un chiffre d&...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Fécondés exempts d&#39;agents pathogènes (SPF) des œufs blancs de poulet Leghorn </td><td> Sunrise Farms (Catskill, État de New York) </td><td></td><td></td></tr><tr><td> Oeuf de poule couveuse </td><td> GQF de fabrication, Savannah, Géorgie </td><td> Modèle-1550 </td><td> Régler à l&#39;humidité de 60% et de 37,5 ° C. </td></tr><tr><td> Tubes capillaires en verre pour </td><td> Mondiale de précision Instrument Inc </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1 ml seringue </td><td> Hamilton Co. Reno, Nevada </td><td> Étanche au gaz 1710 </td><td> Seringue En variante 1ml peut être utilisé aussi bien </td></tr><tr><td> Masterflex silicone (peroxyde) Tube </td><td> Cole-Parmer </td><td> HV-96400-13 </td&gt; <td> Coupez un petit morceau (1 cm) pour attacher l&#39;aiguille de verre à la seringue </td></tr><tr><td> Pince à épiler </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Micromanipulateur </td><td> Mondiale de précision Instrument Inc </td><td> M3301-M3 </td><td></td></tr><tr><td> L&#39;ADN plasmidique </td><td></td><td></td><td> pCAG-GFP a été emprunté par le Dr Connie Cepko </td></tr><tr><td> Fast Green FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Diluez à 0,025% avec du PBS </td></tr><tr><td> Le générateur d&#39;impulsions </td><td> Harvard Apparatus, MA </td><td> BTX ECM 830 </td><td> Générateur d&#39;ondes carrées </td></tr><tr><td> Électrodes </td><td> Harvard Apparatus, MA </td><td> Modèle BTX 514 </td><td> Nos électrodes sont espacées de 3-5 mm en dehors </td></tr><tr><td> Appareil photo numérique monochrome </td><td> Zeiss, en Allemagne </td><td> Unexiocam MRM </td><td></td></tr><tr><td> Microscope à dissection fluorescent </td><td> Leica Microsystems, Allemagne </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Upright Microscope à fluorescence </td><td> Zeiss, en Allemagne </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

Rétine de poulet embryonnaire est un excellent outil pour étudier le développement de la rétine chez les vertébrés supérieurs. Parce que de grande taille et de développement externe, il est relativement très facile de manipuler la rétine de poulet embryonnaire en utilisant l&#39;ADN recombinant / technologie de l&#39;ARN. L&#39;électroporation de l&#39;ADN / ARN des constructions dans la rétine embryonnaire ont un grand avantage pour étudier la régulation des gènes dans la rétine cellules souches / progénitrices au cours du développement de la rétine. Différents types de tests tels que dosage du gène rapporteur, gène sur-expression, le gène abattre (shRNA), etc peut être réalisée en utilisant la technique d&#39;électroporation. Cette vidéo montre ciblé d&#39;injection de la rétine et dans électroporation in ovo dans la rétine de poulet embryonnaire au stade de Hamburger et Hamilton 22-23, ce qui représente environ jour embryonnaire 4 (E4). Ici, nous montrons une réponse rapide et pratique dans lequel technique d&#39;électroporation in ovo un ADN plasmidique qui exprime la protéine fluorescente verte (GFP) comme un marqueur est livré directement dans lapoussin embryonnaire espace sous-rétinien et suivie par des impulsions électriques pour faciliter l&#39;absorption de l&#39;ADN par la rétine cellules souches / progénitrices. La nouvelle méthode d&#39;injection de la rétine et l&#39;électroporation à E4 permet la visualisation de tous les types de cellules rétiniennes, y compris la fin de neurones d&#39;origine 1, qui a été difficile avec la méthode conventionnelle de l&#39;injection et l&#39;électroporation à E1.5 2.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

L&#39;objectif global de cette vidéo est de montrer comment effectuer l&#39;injection ciblée de la rétine et<em&gt; In ovo</emÉlectroporation&gt; de l&#39;ADN / ARN des constructions dans la rétine de poulet embryonnaire au Hamburger et le stade de Hamilton 22-23, ce qui représente environ jour embryonnaire 4 (E4). Cette technique est très utile pour étudier l&#39;expression des gènes, la régulation des gènes, et les changements morphologiques dans le développement de la rétine de poulet.

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 ...

in-ovo-electroporation-in-embryonic-chick-retina

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JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K vues.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. L'objectif global de cette vidéo est de montrer comment effectuer l'injection ciblée de la rétine et In ovo de l'ADN / ARN des constructions dans la rétine de poulet embryonnaire au Hamburger et le stade de Hamilton 22-23, ce qui représente environ jour embryonnaire 4 (E4). Cette technique est très utile pour étudier l'expression des gènes, la régulation des gènes, et les changements morphologiques dans le développ...

Vidéo: In Ovo Electroporation in Embryonic Chick Retina

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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Biologie

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Méthode de coloration Mont anticorps entier de Chick

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

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.

Dosage de l&#39;induction neurale dans l&#39;embryon de poulet

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.

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 ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

La transfection seule cellule dans des embryons de poulet

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...