We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

Toutes les procédures décrites dans cet ouvrage ont été réalisés selon les directives recommandées par la protection des animaux et l&#39;utilisation Comité à l&#39;Université Johns Hopkins. 1. Ahead of Time: préparation d&#39;instruments, réactifs et Plats <ol><li> Préparation des oeufs: incuber des oeufs fertilisés de poulet White Leghorn à 37,5 ° C et 60% d&#39;humidité relative pendant 5 jours dans un incubateur d&#39;œufs. REMARQUE: Un incubateur avec des rocking capacité peut être utile pour la normalisation et la synchronisation du développement embryonnaire, mais bascule est pas strictement nécessaire pour l&#39;issue de ce protocole. </li><li> Le plasmide Préparation: <ol><li> Conception construction plasmidique pour la surexpression ou la régulation négative du gène (par exemple par l&#39;utilisation de la technologie ARNi 1,34) selon les besoins. Inclure un gène rapporteur (tels que EGFP, RFP ou LacZ) dans le produit d&#39;assemblage chaque fois que possible. NOTE: Certains promoteurs couramment utilisés, tels que le CMV, peuvent se taire après le temps. Le Promot CAGer (poulet bêta-actine promoteur CMV activateur) est une très bonne alternative, fournissant une expression efficace à long terme du gène d&#39;intérêt 35-37. </li><li> Préparer une solution de plasmide à une concentration de 1,5 ug / ul dans du PBS stérile. OPTION: Préparer la solution de plasmide à l&#39;avance et de le stocker au congélateur jusqu&#39;au moment d&#39;utiliser. NOTE: Des concentrations plus élevées de plasmide ne conduisent pas à une augmentation significative de l&#39;efficacité, tandis que l&#39;utilisation des concentrations de plasmide moins élevés abaissent l&#39;efficacité de transfection 1. </li></ol></li><li> Électrodes: <ol><li> Pour l&#39;utilisation de l&#39;anode une médaille d&#39;or épaisse basculé électrode (voir le tableau des matériaux spécifiques / Équipement). Essuyer avec 70% d&#39;éthanol. REMARQUE: Isolation de l&#39;électrode à bout l&#39;or, ce qui laisse environ 0,5 mm apparentes, est souhaitable pour minimiser les fuites de courant électrique. Information pour matériau isolant peut être trouvé dans le tableau des matériaux spécifiques. </li><li> Faire une cathode sur un carré de 2,5 mm bboeuf filament, 4,5 mm de large (normalement utilisé dans les extracteurs de micropipette). Avec l&#39;aide de forceps, annuler la boîte carrée et redresser filament en une longue bande. Puis, sous la dissection microscope et en utilisant une règle comme guide, plier le filament dans une forme carrée &#39;U&#39; 3 mm de long à la base et 2 mm de haut sur ​​un côté, de l&#39;autre côté de la longueur restante du filament (Figure 1 , C - D). En utilisant des pinces, tremper l&#39;électrode dans 96% d&#39;éthanol et de la flamme pour stériliser. Ensuite, fixez un fil électrique avec une petite pince crocodile à bras le plus long de l&#39;électrode. </li></ol></li><li> Électroporation Chambre Setup: <ol><li> Préparer la chambre d&#39;électroporation en coupant le couvercle d&#39;un tube de 1,5 ml à centrifuger stérile et en l&#39;apposant, côté plat vers le bas, à un 100 mm boîte de Petri en utilisant du ruban (figure 1E). REMARQUE: un autre récipient sous la forme d&#39;un puits de taille similaire pourrait fonctionner; Toutefois, un couvercle de microtube est facilement accessible et autoclavable. Le puitdoit être suffisant pour abriter une électrode poussin oeil et la cathode grand, mais pas si grand qu&#39;il en résulte une perte de solution de plasmide. </li></ol></li></ol> 2. Ex ovo procédure électroporation <ol><li> Préparation des instruments: <ol><li> Stériliser les outils micro-dissection (2 grosses paires de pinces Moloney courbes pour l&#39;ouverture des coquilles d&#39;oeufs et creusant embryons, 2 paires de fine pointe recourbée pinces Dumont pour enlever RPE, une paire de Bonn dentée pince pour enlever la lentille et vitreuse) en plongeant le conseils dans 96% d&#39;éthanol et enflammées. Champ de dissection propre en utilisant des lingettes trempées dans 70% d&#39;éthanol. Réchauffez une bouteille de solution saline équilibrée de stérile de calcium-magnésium sans Hank (CMF-HBSS) dans un bain d&#39;eau à 37 ° C. Remplissez 100 mm boîtes de Petri stériles à 3/4 avec de chaleureux CMF-HBSS. </li><li> Placez chambre d&#39;électroporation sous un autre microscope de dissection propre et remplir avec 120 pi de solution de plasmide. Connectez électrodes pour appareil d&#39;électroporation, faisantassurer électrode sur mesure est relié au pôle négatif et l&#39;or basculé électrode au pôle positif. </li></ol></li><li> Embryon Collection: <ol><li> Nettoyez les oeufs en les essuyant avec coquilles lingettes humidifiées dans 70% d&#39;éthanol. Maintenir procédure stérile dans le reste du protocole à éviter de transporter la contamination dans les cultures. Utilisation de grandes pinces Moloney courbes ouvrir soigneusement un trou dans la coquille d&#39;oeuf en tapotant doucement sur le bout arrondi d&#39;un œuf (plus de la chambre à air), puis saisir et tirer des morceaux de coquille. </li><li> Avec une autre paire de pinces supprimer membranes et de recueillir embryon en écopant de l&#39;œuf (en faisant attention à ne pas endommager l&#39;embryon). Placez-le dans un plat avec le FMC HBSS. REMARQUE: Recueillir autant d&#39;embryons que nécessaire pour obtenir le nombre de tasses désiré rétiniennes pour électroporation comme spécifié dans 2.4.4 </li><li> Étape critique: stade, les embryons selon Hamburger et Hamilton 38. REMARQUE: Pour obtenir des embryons d&#39;efficacité optimalesdoit être au stade 27 (figure 2A). </li><li> Euthanasier les embryons par décapitation à l&#39;aide de forceps Moloney. </li></ol></li><li> Obtention Coupes de la rétine: <ol><li> En utilisant des pinces de Dumont fines, énucléer attentivement les yeux et de les transférer à un nouveau plat CMF-HBSS. </li><li> A partir de la partie postérieure de chaque œil, à proximité de la tête du nerf optique, et l&#39;aide de deux pinces de Dumont fines, introduire une pointe de chaque paire de pinces entre la rétine neurale et RPE et soigneusement disséquer l&#39;EPR être prudent pour ne pas endommager l&#39;neuronal rétine (figure 2B). NOTE: Le manque de Ca 2+ et Mg 2+ dans la solution aidera le RPE détacher de la rétine neurale plus facilement. </li></ol></li><li> Électroporation: <ol><li> Mettre en place les paramètres de électroporateur de livrer 5 impulsions carrées de 15 Volts, 50 msec et 950 msec intervalle. Place &#39;U&#39; électrode négative en forme de l&#39;intérieur de la chambre d&#39;électroporation (couvercle remplissage à centrifugered avec 120 pi d&#39;une solution contenant le plasmide-). </li><li> L&#39;aide de pincettes Dumont courbes transférer une tasse de la rétine à la chambre d&#39;électroporation (en creusant, pas touche), et le placer dans l&#39;électrode en forme de «U», avec la partie postérieure de la rétine vers le fond de l&#39;électrode et la lentille vers le haut ( Figure 2D). </li><li> Placer l&#39;anode de sorte qu&#39;il touche la partie antérieure de l&#39;oeil, à côté de la lentille, et l&#39;utilisation de la pédale de l&#39;électroporateur délivrer des impulsions électriques. </li><li> Transfert tasse rétinienne électroporation à une nouvelle boîte de Pétri avec FMC-HBSS et répéter la procédure d&#39;électroporation avec chacune des tasses rétiniennes restantes. NOTE: jusqu&#39;à 6 coupes rétiniennes peut être une électroporation avec la même solution de plasmide sans une diminution significative de l&#39;efficacité de transfection. </li><li> Retirez la lentille et le corps vitré de chaque tasse rétinienne électroporation en les saisissant avec une pince à Bonn dentées et en tirant doucement à travers til élève ouverture en gardant l&#39;œil en place avec le dos de la pince à épiler Dumont courbes. </li><li> Passer à la dissociation et la culture des cellules de la rétine électroporation suivant les procédures standard. NOTE: Pour un protocole détaillé sur la dissociation et la culture de cellules rétiniennes chiches réfèrent à Vergara et Canto-Soler, 2012, fichier complémentaire 4 21 Dans ce protocole, les cellules sont ensemencées à faible densité sur des plaques 6 puits polyornithine enduit ou 35. -MM plats (6 x 10 5 cellules / 35 mm de diamètre ainsi ou plat). Le milieu utilisé est un milieu 199 avec de la pénicilline / glutamine, 10% de sérum de veau foetal (FCS), et de l&#39;acide linoléique-BSA à 100 ug / ml. Les cultures sont maintenues dans un 37 ° C humidifié incubateur avec 5% de CO 2. En utilisant cette procédure, 5 - 6 x 10 6 cellules peuvent généralement être obtenus par chaque étape 27 oeil électroporation. NOTE: Dans l&#39;idéal, pas plus de 3 h devrait passer entre la collecte des embryons et le placage de cellules, pour assurer une bonne survie des celluleset minimiser le stress. Un calendrier typique pour l&#39;observation et l&#39;analyse plus approfondie est au bout de 4 jours de culture, car à ce stade, les cellules ont réalisé une bonne différenciation morphologique et moléculaire et la survie est encore élevé. </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. 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=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</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> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

L&#39;étape la plus critique pour le succès de ce protocole est de sélectionner le stade approprié des embryons. Dans les publications précédentes, une série de stades embryonnaires est donnée pour ces cultures, généralement définie par les jours d&#39;incubation ou embryonnaires jours (ED); donc il est généralement supposé que l&#39;utilisation ED ED 5 à 6 embryons donnera des résultats équivalents. Cependant, nous avons constaté que sur scène 27 (ED 5), l&#39;efficacité de transfection sera autour ...

Des cultures de cellules dissociées à partir de rétines embryonnaires de poulet ont été largement utilisés pour étudier les différents aspects de la biologie des cellules de photorécepteur, y compris leur survie 09/02, 12/10 différenciation, la croissance des neurites 13, et plus encore. Les avantages de ce système, mis au point dans les années 1980 par Ruben Adler et collaborateurs et perfectionné par ses et d&#39;autres groupes 14-20, résident dans les caractéristiques intrinsèques de la nana comme un modèle animal 21. La grande taille de l&#39;œil de poulet, même à des stades embryonnaires, fournit de grandes quantités de matériel pour les cultures. En outre, lorsque les cultures sont réalisées en utilisant jour embryonnaire (ED) 5 - 6 rétines, de 55 à 80% de leurs cellules progénitrices différencier en photorécepteurs 14,15,18,22,23, et puisque environ 86% des photorécepteurs de cet animal sont cônes 24, ces cultures sont particulièrement adaptés pour les études portant sur ​​ce type de cellule. Nous avons récemment déveped et caractérisé une technique simple qui permet à haut rendement plasmide transfection des cellules dans ces cultures, élargissant ainsi l&#39;utilité de ce système en facilitant les études de génétique missexpression 1. Le développement de cette technique découle d&#39;un vide dans la littérature scientifique des méthodes qui permettraient un niveau acceptable de transfection pour permettre l&#39;étude de la fonction des gènes d&#39;une manière autonome cellulaire. Ceci est en partie à cause des cultures de neurones primaires sont notoirement difficiles à transfecter 25,26. Certaines des techniques les plus couramment utilisées précédemment disponibles à cet effet inclus méthodes de transfection chimiques tels que la lipofection ou phosphate de calcium, transfection qui se traduisent par des rendements de l&#39;ordre de 3-5% et peuvent exercer une toxicité considérable 27-32. Même si l&#39;utilisation de plasmides avec un système rapporteur enzymatique peut contourner le problème de la mauvaise efficacité de la transfection par l&#39;amplification du signal, ils ne le font pasdiscriminer les effets spécifiques des cellules, et leurs résultats sont basés sur une petite population de cellules qui peuvent ne pas être représentative de l&#39;ensemble. Une autre méthode largement utilisée dans le poussin, infection par le virus RCAS, est applicable uniquement aux cellules proliférantes et ne conviennent donc pas pour cette primaire système de culture de la rétine 33. Dans le protocole actuel yeux chiches embryonnaires sont énucléés à l&#39;étape 27 (ED 5), l&#39;épithélium pigmentaire rétinien (EPR) est enlevée, et la coupe de la rétine est placé dans une chambre d&#39;électroporation rempli d&#39;une solution contenant le plasmide et électroporation en utilisant sur mesure des électrodes, suivie de dissociation de la rétine et de la culture en utilisant des techniques standards 21. Après l&#39;optimisation de cette procédure, nous avons été en mesure de réaliser systématiquement des efficacités de transfection de l&#39;ordre de 22% du nombre total de cellules en culture et 25% au sein de la population de photorécepteur seul, sans compromettre les caractéristiques de survie et la différenciation descultures 1. Ici, nous fournissons un protocole détaillé décrivant toutes les étapes importantes de cette procédure afin d&#39;assurer le succès et la reproductibilité de cette technique.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

Les cultures photoréceptrices enrichi cône provenant de rétines chiches embryonnaires sont devenus un outil indispensable pour les chercheurs du monde entier qui étudient la biologie des neurones de la rétine, en particulier les photorécepteurs. Les applications de ce système vont au-delà de la recherche fondamentale, car ils peuvent facilement être adaptés aux technologies à haut débit pour le développement de médicaments. Toutefois, la manipulation génétique des photorécepteurs de la rétine dans ces cultures est avérée très difficile, ce qui constitue une limitation importante de l&#39;utilité du système. Nous avons récemment développé et validé un plasmide ex ovo technique d&#39;électroporation qui augmente le taux de transfection de cellules de la rétine dans ces cultures par cinq fois par rapport à d&#39;autres protocoles actuellement disponibles 1. Dans cette méthode yeux chiches embryonnaires sont énucléés à l&#39;étape 27, l&#39;EPR est enlevée, et la coupe de la rétine est placé dans une solution contenant le plasmide et électroporation utilisant CUSTOM- facilement construitsélectrodes faites. Les rétines sont ensuite dissociés et cultivées en utilisant des procédures standard. Cette technique peut être appliquée à des études de surexpression, ainsi que la régulation négative de l&#39;expression génique, par exemple via l&#39;utilisation de la technologie d&#39;ARNi induite par le plasmide, la réalisation couramment l&#39;expression du transgène dans 25% de la population ayant une photorécepteur. Le format vidéo de la présente publication rendre cette technologie facilement accessible aux chercheurs dans le domaine, ce qui permet l&#39;étude de la fonction des gènes dans les cultures primaires rétiniennes. Nous avons également inclus des explications détaillées sur les étapes essentielles de cette procédure pour le succès et la reproductibilité.

Nous présentons ici un protocole simple pour le plasmide transfection dans énucléés chiches tasses rétiniennes pour la culture cellulaire dissociée ultérieure. La transfection est réalisée par électroporation en utilisant facile à faire personnalisés électrodes (Figures 1 - 2). Les paramètres décrits dans ce protocole ont été optimisés pour obtenir des efficacités de transfection qui se situent entre 20 et 27% (avec une moyenne de 22%) (Figure 3D). No...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Cultures embryonnaires de cellules rétiniennes chiches constituent des outils précieux pour l&#39;étude de la biologie photorécepteur. Nous avons mis au point une technique de transfert de gène efficace sur la base de plasmide électroporation ex ovo de la rétine antérieure à la culture. Cette technique augmente considérablement les efficacités de transfection sur des protocoles actuellement disponibles, ce qui rend possible une manipulation génétique pour ce système.

Efficient Gene Transfer dans Chick rétines pour cellulaires primaires études sur la culture: Un<em&gt; Ex-ovo</em&gt; Approche d&#39;électroporation

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K vues.  The Johns Hopkins University School of Medicine.  Cultures embryonnaires de cellules rétiniennes chiches constituent des outils précieux pour l'étude de la biologie photorécepteur. Nous avons mis au point une technique de transfert de gène efficace sur la base de plasmide électroporation ex ovo de la rétine antérieure à la culture. Cette technique augmente considérablement les efficacités de transfection sur des protocoles actuellement disponibles, ce qui rend possible une manipulation génétiqu...

Vidéo: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

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

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

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

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimisé<em&gt; Ex-ovo</em&gt; Culture des embryons de poulet aux stades avancés de développement

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

Recherche

Biologie du développement

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Culture de souris embryonnaires cochléaires explants et Gene Transfer par électroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Une approche pour l&#39;évaluation optogénétiques formation des connexions neuronales dans un système de co-culture

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Approche génétique de l&#39;avant pour découvrir les gènes de résistance au stress chez la souris - un écran haute-débit dans les cellules ES

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Culture ex vivo de Chick cervelet tranches et spatialement ciblées électroporation de granules précurseurs de cellules

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Transfert de gènes dans l&#39;organe de poulet Auditory par<em&gt; Dans Ovo</em&gt; Micro-électroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Un roman<em&gt; Ovo Ex</em&gt; Banding Technique d&#39;Alter intracardiaque hémodynamique dans un système embryonnaire de poulet

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

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 filtre Sandwich de papier Submergé Long terme<em&gt; Ovo Ex</em&gt; Time-lapse Imaging of Early Chick Embryons

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 ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR induite par la perte de fonction dans cervelet granules des cellules utilisant In Utero électroporation basée sur un transfert de gènes

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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.

Méthodes in ovo et ex ovo pour étudier le développement de l’oreille interne aviaire

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 ...