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.

NOTA: Tutti i protocolli che impiegano animali vivi deve essere esaminato e approvato da una cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) e deve seguire i metodi per la cura e l&#39;uso di animali da laboratorio ufficialmente approvato. Tutte le dissezioni devono essere eseguiti con tecnica sterile su un banco a flusso laminare pulita. Guanti e una maschera, se desiderato, devono essere indossati durante questa procedura. 1. La dissezione del Ear embrionali di topo interno <ol><li> Impostazione per organo di culture espianto Corti: <ol><li> Sterilizzare la cappa coltura tissutale flusso laminare accendendo la luce UV per circa 30 minuti e disinfettare la superficie con etanolo al 70% prima dell&#39;uso. Inoltre, sterilizzare tutti gli strumenti di dissezione tra cui strumenti di dissezione raffinati e piatti Sylgard rivestite di embrione dissezione tramite autoclave o per immersione in etanolo al 70% per almeno 20 minuti prima dell&#39;uso. Eliminare il 70% di etanolo e lasciare i piatti e gli strumenti per aria secca in un flusso laminare Clea puliton panchina prima dell&#39;uso. </li><li> Preparare Balanced Salt Solution sterile di Hank (HBSS) / (4- (2-idrossietil) -1-acido piperazineethanesulfonic) (HEPES) miscela aggiungendo 10% di 10x HBSS e 0,5% HEPES e regolare il pH a circa 7,2 e sterilizzare il filtro soluzione finale e conservare a 4 ° C. Eseguire tutte le dissezioni di refrigerate soluzione HBSS / HEPES per conservare meglio il tessuto durante tutta la procedura. </li><li> Piatti fondo di vetro a matrice Coat membrana basale aggiungendo 5 ml di freddo (4 ° C), sterile di Dulbecco modificato (DMEM) per un&#39;aliquota di 300 ml di matrice membrana basale in una sterile 15 ml tubo di polipropilene. Mescolare vortexando contenuti e aggiungere 150 microlitri della miscela di matrice DMEM membrana basale al centro di ciascuna piastra di coltura in modo da coprire l&#39;intera cultura ben presente al centro del piatto. Coprire i piatti e conservare in un 37 ° C incubatore per almeno 45 minuti prima dell&#39;uso. </li><li> Versare 4-5 ml refrigerate HBSS in three sterili piatti polistirene Petri e lasciare chiusi nella cappa. </li><li> Preparare mezzo di coltura in una provetta da 15 ml di polipropilene sterile aggiungendo 9 ml di DMEM, 100 ml di integratori N2, 10 g / ml Cipro e 1 ml di FBS. </li></ol></li><li> Isolamento di embrioni: NOTA: In questa procedura per la coltura di espianti cocleari da topi embrionali, raccolta orecchie interne dal giorno embrionale (E) 13 ossa temporali con il giorno dopo la fecondazione essere considerati come E1 9. Il protocollo può essere utilizzato anche con il mouse embrionale orecchio interno a partire dal E12 a E18. <ol><li> Euthanize un mouse a tempo-incinta di CO 2 e sacrificare animali per dislocazione cervicale o appropriati protocolli approvati. Eseguire euthanization dalla cappa coltura tissutale a flusso laminare per mantenere la sterilità in sala coltura di tessuti. </li><li> Posizionare il mouse eutanasia sul tovagliolo di carta con l&#39;addome rivolto verso l&#39;alto e disinfettare l&#39;addome con il 70% di etanolo. </li><li> Aprire la cavità addominale afferrando la pellecon pinze curve con una mano e dell&#39;epidermide recisi e muscolo lungo la linea mediana con le forbici con un&#39;altra mano. Fare due tagli perpendicolari su entrambi i lati e tirare fuori l&#39;utero attentamente sollevando corna uterine bilaterali con una pinza e staccarla da sotto le forbici del tessuto connettivo con. </li><li> Posizionare la catena embrionale in una capsula di Petri contenente soluzione raffreddata dissezione (miscela HBSS / HEPES) e trasferimento al banco pulito a flusso laminare. </li><li> Rimuovere gli embrioni con attenzione di placenta e metterli in una delle piastre sterili contenenti soluzione dissezione. Se la soluzione diventa sanguinosa, spostarli in una nuova piastra di Petri. NOTA: A questo punto, gli embrioni possono essere messo in scena con una guida messa in scena Theiler. </li><li> Decapitare embrioni pizzicando alla regione del collo con un paio di pinze o forbici pulite e posizionare le teste in un piatto fresco Petri contenente soluzione dissezione raffreddata. </li></ol></li><li> La dissezione delle orecchie interne: <ol><li> Luogo one testa embrione in un piatto Sylgard sterile contenente dissezione a freddo </li><li> soluzione. Lavorando sotto un microscopio da dissezione con un ingrandimento di 1,6X ~, immobilizzare la testa dell&#39;embrione posizionando perni Minutien intorno o più vicino alla zona degli occhi (Figura 1A). </li><li> Utilizzando due coppie di pinze sterili rimuovere con attenzione la pelle e aprire il cranio sul lato dorsale lungo la linea mediana (Figura 1B). Rimuovere cervello dalla cavità cranica e orecchie interne situate all&#39;interno ossa temporali possono essere identificate in questa fase (Figura 1C, D). Il rivestimento del vaso sanguigno, che di solito è presente intorno l&#39;orecchio interno, può essere utilizzato come punto di riferimento per identificare l&#39;orecchio interno in questa fase (Figura 1D). </li><li> Sezionare l&#39;orecchio interno dalle ossa temporali posizionando la pinza sotto il tessuto e isolando l&#39;orecchio interno dalla base del cranio e trasferimento in un piatto contenente soluzione di dissezione a freddo (Figura 1E, F). </li></ol></li></ol> 2. Generazione di Organo delle Culture Corti Explant NOTA: In questa fase di sviluppo, il tessuto è cartilagineo e può essere facilmente sezionato con pinze. <ol><li> Orient l&#39;orecchio interno in modo che parte ventrale è rivolto verso l&#39;alto (Figura 2A) e stabilizzare l&#39;orecchio interno inserendo dolcemente la punta di perni Minutien attraverso la porzione vestibolare dell&#39;orecchio interno (Figura 2B). </li><li> Uso pinze ultrafini tagliare aprire la cartilagine sovrastante facendo un&#39;incisione con una estremità di pinze vicino alla finestra ovale e rimuovere la cartilagine dalla coclea (Figura 2C) con attenzione. NOTA: È importante assicurarsi che pinze non sono inserite troppo profondamente nella cartilagine cartilagine sovrastante è talvolta fuso con il condotto cocleare nel qual caso è utile usare pinza chiusa per rilasciare la cartilagine dal condotto cocleare sottostante raschiando sotto la surface della cartilagine. A questo stadio di sviluppo, la spirale cocleare è solo tre quarti di giro in lunghezza. </li><li> Successivamente, esporre dell&#39;epitelio sensoriale posizionando la pinza alla regione preferita del condotto cocleare, sia la base o per lo all&#39;apice, e tirando delicatamente il tetto della coclea (Figura 2D). NOTA: Il tetto della coclea deve essere completamente rimosso in quanto può mascherare dell&#39;epitelio sensoriale ostacolare l&#39;analisi. NOTA: Questa procedura deve essere eseguita delicatamente come è facile da strappare dell&#39;epitelio sensoriale. </li><li> Come ultima fase, rimuovere con attenzione il tessuto connettivo sottostante dall&#39;epitelio sensoriale esposto modo che la base del trapianto cocleare è uniformemente piana. </li><li> Isolare la coclea sezionato dalla parte vestibolare dell&#39;orecchio interno con pinze. Questo può essere eseguito prima o dopo la fase 2.4 (figura 2E). </li><li> Trasferire l&#39;epitelio sensoriale cocleare sezionato in un seminterrato membrane matrix-rivestita cultura e con una paletta sterile 1,5 millimetri (Figura 2F). </li><li> Orient espianto cocleare con la superficie luminale dell&#39;epitelio rivolto verso l&#39;alto e aspirare la membrana basale soluzione di matrice-DMEM per appiattire accuratamente il tessuto e sostituirlo con 150 ml di terreno di coltura fresco aggiungendo delicatamente al piatto. Si deve prestare attenzione per assicurarsi che ogni espianto aderisce bene alla coprioggetto vetro rivestito e non sta galleggiando nel mezzo di coltura. Assicurarsi che la punta della pinza è rivolta verso l&#39;alto, mentre apponendo sul bene di vetro per evitare di danneggiare l&#39;espianto cocleare. </li><li> Trasferire delicatamente il piatto di cultura in un 150 millimetri piatto di coltura sterile e posto in un tessuto culturale incubatore a 37 ° C con il 5% di CO 2 per il periodo di tempo desiderato, di solito 3-6 giorni in vitro (DIV). Dopo 1 DIV, esaminare le culture sotto un microscopio da dissezione per assicurarsi che l&#39;espianto cocleare aderisce bene al piatto di coltura. </li><li> Dopo incubation in vitro per periodi di tempo desiderato, le culture sono trattati per immunoistochimica. NOTA: Le culture espianto di solito vengono incubate per 6 DIV che è equivalente a stadio di sviluppo, P0. Dopo incubazione in vitro, le culture possono essere trattati per altre applicazioni a valle come immunoistochimica (Figura 3B), RT-PCR, ibridazione in situ, e / o Western Blot. </li></ol> 3. Elettroporazione mediata Gene Transfer <ol><li> Configurazione per elettroporazione: <ol><li> Sterilizzare uno 100 millimetri vetro piatto Sylgard rivestite in autoclave o immersione in etanolo al 70% per circa 20 minuti e lasciare asciugare in un banco pulito prima dell&#39;uso. </li><li> Preparare DNA da un vettore di espressione di scelta utilizzando opportuni kit simo o midi-preparazione. I vettori di espressione utilizzati in questo protocollo sono pIRES2-Atoh1.EGFP e pCLIG-NeuroD1.EGFP. La concentrazione finale di DNA deve essere di almeno 1 mg / mL in DNA sterile /Senza RNAse acqua. </li></ol></li><li> Electroporating DNA in espianti cocleari embrionali: <ol><li> Aggiungere 10 ml di soluzione di DNA plasmidico da 100 mm a piatto Sylgard rivestite fresco e trasferire un espianto cocleare completamente sezionato (ottenuto dalla fase 2.5) nella soluzione di DNA. </li><li> Con superficie luminale dell&#39;epitelio rivolto verso l&#39;alto, inclinare la coclea leggermente in modo che sia perpendicolare al piano del piatto. </li><li> Collocare le piastre di elettrodi su entrambi i lati della coclea con paddle negativo (catodo) verso l&#39;epitelio sensoriale e la pala positivo (anodo) adiacente alla base della coclea. NOTA: Poiché DNA è caricato negativamente, migra da negativo a elettrodo positivo e inserendo l&#39;estremità del catodo della sonda adiacente dell&#39;epitelio sensoriale; il DNA principalmente passare attraverso la superficie dell&#39;epitelio sensoriale. </li><li> Utilizzando elettroporatore, consegnare 9 a 10 impulsi di 24 mV, 30 msec durata dell&#39;impulso con l&#39;interruttore a pedale su tegli elettroporatore e aggiungere 100 ml di terreno di coltura caldo alla coclea elettroporate. </li><li> Ripetere i passaggi 3.2.1-4 per tutti gli espianti cocleari e per il DNA di tutti i geni di interesse e di trasferire il coclee elettroporate nel piatto di matrice rivestite membrana basale per la placcatura (passo 2,7). Culture tutte le coclee elettroporate a 37 ° C, 5% CO 2 incubatore umidificato per lunghezze desiderate di tempo che sarà poi elaborati per immunocitochimica. NOTA: La coclea da un&#39;intera cucciolata di CD1 mouse (circa 8-12 cuccioli) può essere isolato, microdissezione, elettroporate e placcato in &lt;4 ore. </li></ol></li></ol> 4. Analisi delle Culture Cochlear Explant NOTA: Le colture vengono incubate per 6 solito DIV che è equivalente a stadio di sviluppo, P0. Dopo incubazione in vitro, le culture sono fissi e trattati per immunocitochimica. <ol><li> Dopo lunghezza desiderata di incubation, rimuovere terreno di coltura e rapidamente risciacquare l&#39;espianto cocleare in soluzione salina tamponata con fosfato (PBS) e incubare in 4% paraformaldeide (preparato in PBS) per 15 minuti a temperatura ambiente. </li><li> Lavare il fissativo aggiungendo PBS e sciacquare in PBS per tre volte per 15 min. </li><li> Permeabilize con PBS-T (PBS + 0,5% Tween) per 30 minuti prima di bloccare con PBS-T contenente siero di capra 10%. </li><li> Incubare in soluzione anticorpo primario contenente anticorpo primario in PBS-T con siero di capra 1% notte a 4 ° C. </li><li> Lavare la coclea tre volte a temperatura ambiente utilizzando PBS-T 15 min ciascuna prima di aggiungere anticorpi secondari coniugati Alexa appropriate. </li><li> Dal momento che l&#39;espianto cocleare è colta sul vetrino di vetro può essere gestito individualmente isolando il vetrino dal piatto e montaggio su una diapositiva. Ciò può essere ottenuto immergendo il piatto di coltura in OS30 solvente per almeno 1 ora a temperatura ambiente. Utilizzando la lama di rasoio sterile, staccare il vetrino dal dish e montare su un vetrino e visualizzare sotto un microscopio a fluorescenza. </li></ol>

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No conflicts of interest declared.

Tutte le cellule all&#39;interno del labirinto membranoso dell&#39;orecchio interno del mouse inclusi sensoriale, nonsensory ei neuroni gangliari a spirale sono tutti derivati ​​dallo placodally di derivazione otocyst adiacente al romboencefalo della ectoderma, intorno E8 10-14. Al E11, la regione ventrale del otocyst estende a formare il dotto cocleare e lo sviluppo continua, un gruppo di cellule epiteliali all&#39;interno della coclea, così come in altre regioni del otocyst, diventano specificato come ...

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.

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	<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>
			<td style="width:161px;">26002-15</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dumont # 5 Forceps</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Phosphate Buffered Saline</td>
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			<td dir="LTR" style="width:161px;">10010-023</td>
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			<td dir="LTR" style="width:192px;">Southern Biotech</td>
			<td dir="LTR" style="width:161px;">0100-01</td>
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			<td>Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6791</td>
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			<td height="43" style="height:43px;width:316px;">Myosin 7a</td>
			<td style="width:192px;">Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6790</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.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

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 Views.  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. 

Video: 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.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

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

Ricerca

Biologia dello sviluppo

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.

Cultura e co-coltura di mouse ovaie e follicoli ovarici

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 elettroporazione e Organotipica Fetta Cultura del mouse ippocampale 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.

Differenziamento neurale delle cellule staminali embrionali di topo in siero privo monostrato Cultura

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.

Espianti di retina intero da pollo embrioni per trattamenti di elettroporazione e Agenti chimici

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.

Gene Transfer nel pollo Auditory Organ<em&gt; In Ovo</em&gt; Micro-elettroporazione

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.

Cultura di adulti transgenici Zebrafish retina espianti per live-cell imaging per Multiphoton Microscopia

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.

Dissezione e cultura del rene embrionale del mouse

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-mediata di perdita di funzione di analisi in granuli cerebellari cellule utilizzando nell'Utero basati su elettroporazione trasferimento del Gene

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.

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

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