We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

REMARQUE: Tous les protocoles utilisant des animaux vivants doit être examiné et approuvé par un comité de protection des animaux et l&#39;utilisation institutionnelle (IACUC) et doit suivre officiellement méthodes pour le soin et l&#39;utilisation des animaux de laboratoire approuvé. Tous les dissections doivent être effectuées en utilisant une technique stérile sur un banc à flux laminaire propre. Gants et un masque, si vous le souhaitez, doivent être portés pendant cette procédure. 1. Dissection de l&#39;oreille de la souris embryonnaires intérieure <ol><li> Mise en place pour orgue des cultures d&#39;explants Corti: <ol><li> Stériliser la hotte à flux laminaire de culture de tissus en activant la lumière UV pendant environ 30 min et désinfecter la surface avec 70% d&#39;éthanol avant utilisation. En outre, stériliser tous les instruments de dissection y compris les outils de dissection fine et plats Sylgard revêtus pour la dissection embryon par autoclave ou par trempage dans 70% d&#39;éthanol pendant au moins 20 min avant utilisation. Verser 70% d&#39;éthanol et permettre aux plats et instruments à l&#39;air dans un flux laminaire propre CLEAn banc avant utilisation. </li><li> Préparer une solution saline équilibrée de Hank stérile (HBSS) / (4- (2-hydroxyéthyl) -1-piperazineethanesulfonic) mélange (HEPES) en ajoutant 10% de HBSS 10x et 0,5% de HEPES et ajuster le pH à environ 7,2 et stériliser le filtre solution finale et conserver à 4 ° C. Effectuer toutes les dissections en réfrigérés solution HBSS / HEPES pour mieux préserver le tissu pendant toute la procédure. </li><li> plats à fond de verre-manteau avec matrice de membrane basale par addition de 5 ml de milieu Eagle modifié de froid (4 ° C) stérile Dulbecco (DMEM) à une portion aliquote de 300 ul de matrice de membrane basale dans un tube de 15 ml en polypropylene stérile. Mélanger au vortex le contenu et ajouter 150 ul de la membrane basale mélange DMEM-matrice au centre de chaque boîte de culture de sorte qu&#39;il couvre l&#39;ensemble du puits de culture présent dans le centre du plat. Couvrez les plats et les conserver dans un incubateur à 37 ° pendant au moins 45 min avant utilisation. </li><li> Verser 4-5 ml réfrigérés HBSS en eree plats polystyrène de Pétri stérile et le laisser fermé dans la hotte. </li><li> Préparer un milieu de culture dans un tube de 15 ml en polypropylene stérile en ajoutant 9 ml de DMEM, 100 ul de suppléments N2, 10 g / ml Cipro et 1 ml de FBS. </li></ol></li><li> Isolement des embryons: NOTE: Dans cette procédure pour la culture des explants de souris embryonnaires cochléaires, oreille interne récolte du jour embryonnaire (E) 13 os temporaux avec le jour après la fécondation étant considérés comme E1 9. Le protocole peut également être utilisé avec l&#39;oreille interne de souris embryonnaire débutant à partir de E12 à E18. <ol><li> Euthanasier une souris chronométré-enceinte de CO 2 et de sacrifier des animaux par dislocation cervicale ou protocoles approuvés appropriées. Effectuer l&#39;euthanasie loin de la hotte à flux laminaire de culture de tissus pour maintenir la stérilité dans la chambre de culture de tissu. </li><li> Placez la souris euthanasiés sur une serviette en papier avec de l&#39;abdomen vers le haut et désinfecter abdomen avec 70% d&#39;éthanol. </li><li> Ouvrez la cavité abdominale en saisissant la peauavec une pince incurvés avec une main et de l&#39;épiderme et le muscle coupées le long de la ligne médiane à l&#39;aide de ciseaux avec une autre main. Faites deux coupes perpendiculaires des deux côtés et retirez soigneusement l&#39;utérus en soulevant cornes utérines bilatérales avec une pince et le détacher de dessous des ciseaux de tissu conjonctif à l&#39;aide. </li><li> Placez la chaîne embryonnaire dans une boîte de Pétri contenant une solution glacée de dissection (mélange HBSS / HEPES) et transfert à l&#39;banc propre à flux laminaire. </li><li> Retirer soigneusement embryons à partir de placenta et les placer dans une des boîtes de Pétri stériles contenant une solution de dissection. Si la solution devient sanglante, les déplacer vers une nouvelle boîte de Pétri. NOTE: A ce stade, les embryons peuvent être mis en scène à l&#39;aide d&#39;un guide de mise en scène Theiler. </li><li> Décapitez embryons par pincement à la région du cou avec une paire de pinces propres ou des ciseaux et placer les têtes dans un plat frais Petri contenant une solution de dissection froide. </li></ol></li><li> Dissection de l&#39;oreille interne: <ol><li> Lieu otête de l&#39;embryon ne dans un plat Sylgard stérile contenant dissection froide </li><li> solution. Travailler sous un microscope de dissection à un grossissement de 1.6X ~, immobiliser la tête de l&#39;embryon en plaçant épingles minuties autour ou près de la région de l&#39;œil (figure 1A). </li><li> L&#39;utilisation de deux paires de pinces stériles retirer soigneusement la peau et ouvrir le crâne sur la face dorsale le long de la ligne médiane (figure 1B). Retirer le cerveau de la cavité crânienne et les oreilles intérieures situées dans os temporaux peuvent être identifiés à ce stade (figure 1C, D). La paroi de vaisseau sanguin, ce qui est habituellement présent dans l&#39;oreille interne, peut être utilisé comme un point de repère pour l&#39;identification de l&#39;oreille interne à ce stade (figure 1D). </li><li> Disséquer l&#39;oreille interne des os temporaux en plaçant la pince sous le tissu et l&#39;isolement de l&#39;oreille interne de la base du crâne et le transfert à un nouveau plat contenant une solution de dissection froide (figure 1E, F). </li></ol></li></ol> 2. Génération d&#39;organes des cultures d&#39;explants Corti NOTE: A ce stade de développement, le tissu est cartilagineux et peut facilement être disséqué en utilisant une pince. <ol><li> Orientez le oreille interne afin que la face ventrale est orientée vers le haut (figure 2A) et de stabiliser l&#39;oreille interne en insérant doucement l&#39;extrémité pointue de broches minuties à travers la partie vestibulaire de l&#39;oreille interne (figure 2B). </li><li> Aide d&#39;une pince ultrafines coupés ouvrir le cartilage sus-jacente en faisant une incision en utilisant une extrémité de forceps près de la fenêtre ovale et retirez soigneusement le cartilage de la cochlée (figure 2C). NOTE: Il est important de se assurer que le forceps ne sont pas insérés trop profondément dans le cartilage que le cartilage recouvrant est parfois fusionné avec le canal cochléaire dans ce cas, il est utile d&#39;utiliser des pinces fermées pour libérer le cartilage du conduit cochléaire sous-jacent par grattage en dessous de la surface du cartilage. A ce stade de développement, la spirale cochléaire est à seulement trois quarts de tour en longueur. </li><li> Ensuite, exposer l&#39;épithélium sensoriel en plaçant les pinces à la région préférée du conduit cochléaire, la base ou au sommet très, et en tirant doucement sur ​​le toit de la cochlée (figure 2D). NOTE: Le toit de la cochlée doit être enlevée complètement, car elle peut masquer l&#39;épithélium sensoriel entraver l&#39;analyse. NOTE: Cette procédure doit être fait doucement car il est facile à déchirer l&#39;épithélium sensoriel. </li><li> Comme dernière étape, retirer délicatement le tissu conjonctif sous-jacent de l&#39;épithélium sensoriel exposés, afin que la base de l&#39;expiant cochléaire est uniformément plat. </li><li> Isoler la cochlée disséqué de la partie vestibulaire de l&#39;oreille interne en utilisant une pince. Ceci peut être réalisé avant ou après l&#39;étape 2.4 (figure 2E). </li><li> Transférer l&#39;épithélium sensoriel cochléaire disséqué dans un membra du sous-solne matrice revêtu puits de culture en utilisant un scooper stérile de 1,5 mm (figure 2F). </li><li> Orientez le explant cochléaire avec la surface luminale de l&#39;épithélium vers le haut et aspirer la solution de la membrane basale matrice DMEM pour aplatir soigneusement le tissu et le remplacer par 150 pi de milieu de culture frais en ajoutant doucement pour le plat. Des précautions doivent être prises pour se assurer que chaque explant adhère bien à la lamelle de verre revêtue et ne flotte pas dans le milieu de culture. Assurez-vous que la pointe de la pince est dirigé vers le haut tout en apposant sur le bien de verre pour éviter d&#39;endommager l&#39;expiant cochléaire. </li><li> Transférer doucement la boîte de culture dans une boîte de culture de 150 mm stérile et placer dans un incubateur de culture de tissus à 37 ° C avec 5% de CO 2 pour la période de temps désirée, généralement 3-6 jours in vitro (DIV). Après une DIV, examiner les cultures sous un microscope à dissection pour se assurer que l&#39;expiant cochléaire a ainsi adhéré à la boîte de culture. </li><li> Après encubation in vitro pour les longueurs souhaitées de temps, les cultures sont traitées pour l&#39;immunohistochimie. NOTE: Les cultures d&#39;explants sont généralement incubées pendant 6 DIV qui est équivalent au stade de développement, P0. Après une incubation in vitro, les cultures peuvent être traitées pour d&#39;autres applications en aval tels que l&#39;immunohistochimie (Figure 3B), RT-PCR, l&#39;hybridation in situ, et / ou Western blot en. </li></ol> 3. L&#39;électroporation Gene Transfer médiée <ol><li> Mise en place pour l&#39;électroporation: <ol><li> Stériliser une boîte de 100 mm revêtu de verre SYLGARD par autoclavage ou par trempage dans 70% d&#39;éthanol pendant environ 20 min et laisser sécher à l&#39;air dans un banc propre avant de l&#39;utiliser. </li><li> Préparer l&#39;ADN d&#39;un vecteur d&#39;expression de choix en utilisant des kits de maxi- ou Midi-prep appropriées. Les vecteurs d&#39;expression utilisés dans ce protocole sont pIRES2-Atoh1.EGFP et pCLIG-NeuroD1.EGFP. La concentration finale de l&#39;ADN devrait être d&#39;au moins 1 ug / ul dans l&#39;ADN stérile /RNase-free de l&#39;eau. </li></ol></li><li> Électroporation de l&#39;ADN dans des explants embryonnaires cochléaires: <ol><li> Ajouter 10 ul de solution d&#39;ADN plasmidique à une cuvette revêtue Sylgard 100 mm frais et transférer un explant cochléaire entièrement disséqué (obtenu à l&#39;étape 2.5) dans la solution d&#39;ADN. </li><li> Avec surface luminale de l&#39;épithélium vers le haut, incliner légèrement la cochlée de sorte qu&#39;il est perpendiculaire au plan du plat. </li><li> Placer les palettes d&#39;électrodes de chaque côté de la cochlée à palette négative (cathode) vers l&#39;épithélium sensoriel et la palette positive (anode) située adjacente à la base de la cochlée. NOTE: L&#39;ADN est chargé négativement, il migre de négative à l&#39;électrode positive et en plaçant l&#39;extrémité de cathode de la sonde adjacent à l&#39;épithélium sensoriel; l&#39;ADN sera surtout passer par la surface de l&#39;épithélium sensoriel. </li><li> Utilisation électroporateur, livrer 9-10 impulsions de 24 mV, 30 durée d&#39;impulsion ms avec le commutateur de pédale sur til électroporateur, puis ajouter 100 ul de milieu de culture chaude à la cochlée électroporation. </li><li> Répétez les étapes 3.2.1-4 pour tous les explants cochléaires et de l&#39;ADN de tous les gènes d&#39;intérêt et de transférer la cochlée électroporation dans le plat de la matrice revêtu membrane basale pour le placage (étape 2.7). Culture tout le électroporé dans une cochlée 37 ° C, 5% de CO 2 incubateur humidifié pour des longueurs désirées de temps qui seront ensuite traités par immunocytochimie. NOTE: La cochlée d&#39;une portée entière de souris CD1 (environ 8 à 12 chiots) peut être isolé, microdissection, électroporation et plaqué dans &lt;4 h. </li></ol></li></ol> 4. Analyse des cultures d&#39;explants Cochlear REMARQUE: Les cultures sont incubées pendant 6 généralement DIV qui est équivalent au stade de développement, P0. Après une incubation in vitro, les cultures sont fixées et traitées pour immunocytochimie. <ol><li> Après longueur désirée de incubation, retirer le milieu de culture et rapidement rincer l&#39;explant cochléaire dans une solution saline tamponnée au phosphate (PBS) et incuber à 4% de paraformaldehyde (préparé dans du PBS) pendant 15 min à température ambiante. </li><li> Laver le fixateur en ajoutant du PBS et rincer trois fois dans du PBS pendant 15 min. </li><li> Perméabiliser en utilisant du PBS-T (PBS + 0,5% de Tween) pendant 30 min avant le blocage avec du PBS-T contenant du sérum de chèvre à 10%. </li><li> Incuber dans une solution d&#39;anticorps primaire contenant l&#39;anticorps primaire dans du PBS-T avec du sérum de chèvre à 1% pendant une nuit à 4 ° C. </li><li> Laver trois fois la cochlée à la température ambiante en utilisant du PBS-T 15 min avant d&#39;ajouter l&#39;anticorps secondaire approprié conjugué Alexa. </li><li> Depuis l&#39;explant est cultivé cochléaire sur lamelle de verre, il peut être manipulé individuellement par l&#39;isolement de la lamelle couvre-objet et de la boîte de montage sur une lame. 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No conflicts of interest declared.

Toutes les cellules à l&#39;intérieur du labyrinthe membraneux de l&#39;oreille interne de souris comprenant sensorielle, non sensorielle et les neurones du ganglion spiral sont toutes dérivées du dérivé placodally otocyste situé à côté du cerveau postérieur de l&#39;ectoderme, autour de 10-14 E8. A E11, la région ventrale du otocyste se étend pour former le conduit cochléaire et que le développement se poursuit, un groupe de cellules épithéliales dans la cochlée, ainsi que dans d&#39;autres...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HBSS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:161px;">14065-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HEPES</td>
			<td>Gibco</td>
			<td style="width:161px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dulbecco&#8217;s Modified Medium</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">12430-054</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fetal Bovine Serum</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10082</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">N-2 Supplement (100X)</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Ciprofloxacin Hydrochloride</td>
			<td dir="LTR" style="width:192px;">Cellgro</td>
			<td dir="LTR" style="width:161px;">61-277-RF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Dish 60mm</td>
			<td dir="LTR" style="width:192px;">Kimble Chase</td>
			<td dir="LTR" style="width:161px;">23062-6015/23064-6015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Glass Dish 100 mm</td>
			<td dir="LTR">Kimble Chase</td>
			<td style="width:161px;">23064-10015/23062-10015</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Minutien Pins</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
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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 describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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 ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K vues.  Medical University of South Carolina, College of Medicine. 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. 

Vidéo: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

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

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

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 ...

Recherche

Biologie du développement

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Culture et co-culture de souris ovaires et follicules ovariens

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero L&#39;électroporation et organotypiques Slice culture de souris hippocampe Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Différenciation neurale des souris de cellules souches embryonnaires dans le sérum sans culture monocouche

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Explants rétiniens entières de poulet embryons pour Traitements électroporation et réactif chimique

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

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. ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Culture des adultes Zebrafish Retinal explants Transgenic pour des cellules vivantes par imagerie multiphotonique Microscopie

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissection et culture du rein embryonnaire de la souris

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

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 ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolement et différenciation des myoblastes primaires des explantations de muscle squelettique de souris

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...