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: Todos los protocolos que utilizan animales vivos debe ser revisado y aprobado por el Cuidado y Uso de un Comité Institucional Animal (IACUC) y debe seguir los métodos para el cuidado y uso de los animales de laboratorio homologado. Todas las disecciones se deben realizar utilizando una técnica estéril en un banco de flujo laminar limpio. Guantes y una máscara, si se desea, se deben usar durante este procedimiento. 1. La disección de la Oreja embrionarias de ratón Interior <ol><li> Configuración para órgano de cultivos de explantes Corti: <ol><li> Esterilizar el flujo laminar campana de cultivo de tejido mediante la activación de la luz UV durante aproximadamente 30 min y desinfectar la superficie con etanol al 70% antes de su uso. Además, esterilizar todos los instrumentos de disección, incluyendo herramientas de disección finas y platos recubiertos con Sylgard para la disección de embriones a través de autoclave o por inmersión en etanol al 70% durante al menos 20 min antes del uso. Verter 70% de etanol y permitir que los platos y los instrumentos se sequen al aire en un flujo laminar limpio CLEAn banco antes de su uso. </li><li> Preparar solución salina equilibrada de Hank estéril (HBSS) / (4- (2-hidroxietil) -1-piperazinetanosulfónico) (HEPES) mediante la adición de mezcla de 10% de HBSS 10x y 0,5% HEPES y ajustar el pH a aproximadamente 7,2 y esterilizar el filtro solución definitiva y se almacena a 4 ° C. Llevar a cabo todas las disecciones en solución HBSS / HEPES enfriado para conservar mejor el tejido durante todo el procedimiento. </li><li> Platos de fondo de cristal Escudo con matriz de membrana basal mediante la adición de 5 ml de medio Eagle modificado frío (4 ° C) estéril de Dulbecco (DMEM) a una alícuota de 300 l de matriz de membrana basal en un tubo de polipropileno de 15 ml estéril. Mezclar mediante agitación el contenido y añadir 150 l de la mezcla de la matriz de la membrana basal-DMEM al centro de cada placa de cultivo de modo que cubra la totalidad del pocillo de cultivo presente en el centro del plato. Cubra los platos y se guardan en una incubadora a 37ºC durante al menos 45 minutos antes de su uso. </li><li> Vierta 4-5 ml refrigerados en HBSS ªree placas de Petri de poliestireno estériles y deje cerrada en la campana. </li><li> Preparar medio de cultivo en un tubo de 15 ml de polipropileno estéril mediante la adición de 9 ml de DMEM, 100 l de suplementos de N2, 10 g / ml Cipro y 1 ml de FBS. </li></ol></li><li> Aislamiento de embriones: NOTA: En este procedimiento para el cultivo de explantes cocleares de embriones de ratones, oídos internos cosecha desde el día embrionario (E) 13 huesos temporales con el día después de la fecundación siendo considerados como E1 9. El protocolo también se puede utilizar con el ratón embrionario oídos internos a partir de E12 a E18. <ol><li> La eutanasia a un ratón oportuna embarazada de CO 2 y el sacrificio de los animales por dislocación cervical o protocolos aprobados apropiados. Llevar a cabo la eutanasia de distancia de flujo laminar campana de cultivo de tejido para mantener la esterilidad en la sala de cultivo de tejidos. </li><li> Coloque la eutanasia del ratón sobre una toalla de papel con abdomen hacia arriba y desinfectar abdomen con etanol al 70%. </li><li> Abra la cavidad abdominal por el acaparamiento de la pielcon pinzas curvas con una mano y la epidermis cortadas y los músculos a lo largo de la línea media con la tijera en la otra mano. Hacer dos cortes perpendiculares en ambos lados y tire del útero cuidadosamente levantando cuernos uterinos bilaterales con fórceps y separarla de debajo de las tijeras de tejido conectivo usando. </li><li> Coloque la cadena de embriones en una placa de Petri que contiene solución enfriada disección (mezcla HBSS / HEPES) y traslado al flujo laminar banco limpio. </li><li> Retire cuidadosamente embriones a partir de placenta y colocarlos en una de las placas de Petri estériles que contienen solución de disección. Si la solución se vuelve sangrienta, moverlos a una nueva placa de Petri. NOTA: En este punto, los embriones pueden ser realizaron utilizando una guía de puesta en escena Theiler. </li><li> Decapitar embriones pellizcando en la región del cuello con un par de fórceps o tijeras limpias y colocar las cabezas en una placa de Petri fresco que contenía solución de disección fría. </li></ol></li><li> La disección de los oídos internos: <ol><li> Lugar ocabeza embrión ne en un plato Sylgard estéril que contiene disección fría </li><li> solución. Trabajando bajo un microscopio de disección con un aumento de ~ 1.6X, inmovilizar la cabeza de embriones mediante la colocación de pasadores Minutien alrededor o cerca de región de los ojos (Figura 1A). </li><li> El uso de dos pares de pinzas estériles retire con cuidado la piel y abrir el cráneo en el lado dorsal a lo largo de la línea media (Figura 1B). Retire cerebro de la cavidad craneal y oídos internos ubicados dentro huesos temporales pueden ser identificados en esta etapa (Figura 1 C, D). El revestimiento del vaso sanguíneo, que normalmente está presente alrededor de los oídos internos, se puede utilizar como un punto de referencia para identificar el oído interno en esta fase (Figura 1D). </li><li> Diseccionar el oído interno de los huesos temporales mediante la colocación de las pinzas debajo del tejido y aislando el oído interno desde la base del cráneo y la transferencia a un nuevo plato que contiene la solución de disección fría (Figura 1E, F). </li></ol></li></ol> 2. Generación de Órganos de las Culturas Corti explantes NOTA: En esta etapa de desarrollo, el tejido cartilaginoso es y puede ser diseccionado fácilmente utilizando fórceps. <ol><li> Oriente el oído interno de manera que el lado ventral esté hacia arriba (Figura 2A) y estabilizar el oído interno mediante la inserción suavemente el extremo afilado de pasadores Minutien través de la porción vestibular del oído interno (Figura 2B). </li><li> Utilizando unas pinzas ultrafinas cortar el cartílago suprayacente al hacer una incisión utilizando un extremo de fórceps cerca de la ventana oval y cuidadosamente retirar el cartílago de la cóclea (Figura 2C). NOTA: Es importante asegurarse de que los fórceps no se insertan demasiado profundamente en el cartílago como el cartílago suprayacente a veces se fusiona con el conducto coclear en cuyo caso es útil usar unas pinzas cerradas para liberar el cartílago del conducto coclear subyacente por raspado por debajo de la surface del cartílago. En esta etapa de desarrollo, la espiral coclear es sólo tres cuartos de vuelta de longitud. </li><li> A continuación, exponer el epitelio sensorial mediante la colocación de las pinzas en la región preferida del conducto coclear, ya sea la base o en el mismo ápice, y tirando suavemente el techo de la cóclea (Figura 2D). NOTA: El techo de la cóclea debe ser completamente eliminado, ya que puede enmascarar el epitelio sensorial dificultando el análisis. NOTA: Este procedimiento debe hacerse con cuidado, ya que es fácil de rasgar el epitelio sensorial. </li><li> Como último paso, retirar cuidadosamente el tejido conectivo subyacente del epitelio sensorial expuesto de manera que la base del explante coclear es uniformemente plana. </li><li> Aislar la cóclea disecados de la porción vestibular del oído interno con unas pinzas. Esto se puede realizar antes o después de la etapa 2.4 (Figura 2E). </li><li> Transferir el epitelio sensorial coclear disecado en una membra sótanomatriz recubierta ne cultura bien usando un 1.5 mm cuchara estéril (Figura 2F). </li><li> Oriente el explante coclear con la superficie luminal del epitelio hacia arriba y aspirar la solución de matriz-DMEM membrana basal para aplanar cuidadosamente el tejido y reemplazarla con 150 l de medio de cultivo fresco añadiendo suavemente al plato. Se debe tener cuidado para asegurarse de que cada explante se adhiere bien a la cubreobjetos de vidrio recubierto y no está flotando en el medio de cultivo. Asegúrese de que la punta de las pinzas se señaló arriba mientras que la colocación de al pozo de vidrio para evitar dañar el explante coclear. </li><li> Transferir con cuidado la placa de cultivo en un 150 mm placa de cultivo estéril y colocar en una incubadora de cultivo de tejidos a 37 ° C con 5% de CO 2 para el período de tiempo deseado, generalmente 3-6 días in vitro (DIV). Después de 1 DIV, examinar las culturas bajo un microscopio de disección para asegurarse de que el explante coclear se ha adherido bien a la placa de cultivo. </li><li> Después ende incubación in vitro para longitudes de tiempo deseado, las culturas se procesan para inmunohistoquímica. NOTA: Los cultivos de explantes por lo general se incuban durante 6 DIV que es equivalente a la etapa de desarrollo, P0. Después de la incubación in vitro, los cultivos se pueden procesar para más aplicaciones posteriores como inmunohistoquímica (Figura 3B), RT-PCR, hibridación in situ, y / o Western blot. </li></ol> 3. La electroporación mediada por transferencia génica <ol><li> Configuración para la electroporación: <ol><li> Esterilizar uno 100 mm plato de vidrio recubiertos con Sylgard en autoclave o por inmersión en etanol al 70% durante aproximadamente 20 min y dejar secar al aire en un banco limpio antes de su uso. </li><li> Preparar el ADN de un vector de expresión de elección utilizando kits maxi- o midi-prep apropiados. Los vectores de expresión utilizados en este protocolo son pIRES2-Atoh1.EGFP y pCLIG-NeuroD1.EGFP. La concentración final de ADN debe ser de al menos 1 g / l en el ADN estéril /RNasa libre de agua. </li></ol></li><li> Electroporación de ADN en explantes cocleares embrionarias: <ol><li> Añadir 10 l de solución de ADN de plásmido a un 100 mm plato de Sylgard recubierto fresco y transferir un explante coclear totalmente diseccionado (obtenido en la etapa 2.5) en la solución de ADN. </li><li> Con la superficie luminal del epitelio hacia arriba, incline la cóclea ligeramente de modo que es perpendicular al plano del plato. </li><li> Coloque las paletas de electrodos a ambos lados de la cóclea con paleta negativo (cátodo) hacia el epitelio sensorial y la pala positivo (ánodo) situado adyacente a la base de la cóclea. NOTA: Dado que el ADN está cargado negativamente, que migra de negativo a electrodo positivo y colocando el extremo del cátodo de la sonda adyacente al epitelio sensorial; el ADN mayoría pasará a través de la superficie del epitelio sensorial. </li><li> Utilizando electroporador, entregar 9 a 10 pulsos de 24 mV, 30 ms duración del pulso con el interruptor de pedal en tél electroporador, y luego añadir 100 l de medio de cultivo cálido a la cóclea a electroporación. </li><li> Repita los pasos 3.2.1-4 para todos los explantes cocleares y de ADN de todos los genes de interés y transferir el cochleae electroporación en el plato matriz recubiertos con membrana basal para chapado (paso 2.7). Cultura toda la cochleae electroporación en un 37 ° C, 5% de CO 2 humidificado incubadora para longitudes deseadas de tiempo que luego serán procesados ​​por inmunocitoquímica. NOTA: La cóclea de una camada entera de ratón CD1 (aproximadamente 8-12 crías) se puede aislar, microdissected, electroporación y chapada en &lt;4 hr. </li></ol></li></ol> 4. Análisis de las Culturas Coclear explantes NOTA: Los cultivos se incubaron durante por lo general 6 DIV que es equivalente a la etapa de desarrollo, P0. Después de la incubación in vitro, los cultivos se fijaron y procesaron para inmunocitoquímica. <ol><li> Después de longitud deseada del incubation, eliminar el medio de cultivo y rápidamente enjuagar el explante coclear en solución salina tamponada con fosfato (PBS) y se incuban en 4% de paraformaldehído (preparado en PBS) durante 15 min a temperatura ambiente. </li><li> Lave el fijador mediante la adición de PBS y enjuague en PBS tres veces durante 15 minutos. </li><li> Permeabilizar utilizando PBS-T (PBS + 0,5% Tween) durante 30 min antes de bloquear con PBS-T que contiene 10% de suero de cabra. </li><li> Incubar en solución de anticuerpo primario que contiene el anticuerpo primario en PBS-T con 1% de suero de cabra durante la noche a 4 ° C. </li><li> Lavar la cóclea tres veces a temperatura ambiente usando PBS-T 15 min cada uno antes de la adición de anticuerpos secundarios conjugados Alexa-apropiados. </li><li> Desde el explante coclear es cultivaron en cubreobjetos de vidrio puede ser manejado individualmente aislando el cubreobjetos de la placa de montaje y en una diapositiva. Esto se puede lograr por remojo la placa de cultivo en OS30 disolvente durante al menos 1 hora a temperatura ambiente. Uso de la hoja de afeitar estéril, extraiga el cubreobjetos de la dish y montar en un portaobjetos y visualizar bajo un microscopio fluorescente. </li></ol>

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No conflicts of interest declared.

Todas las células dentro de la laberinto membranoso del oído interno de ratón incluyendo sensorial, no sensorial y las neuronas del ganglio espiral se derivan de placodally derivados de otocyst situado adyacente a la parte posterior del cerebro del ectodermo, alrededor de 10-14 E8. En E11, la región ventral de la otocyst se extiende para formar el conducto coclear y como sigue el desarrollo, un grupo de células epiteliales dentro de la cóclea, así como en otras regiones del otocyst, convertido especifi...

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>
			<td dir="LTR" style="width:161px;">11251-10</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 de embriones de ratón explantes cocleares y Gene Transfer por electroporación

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 vistas.  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.

Transferencia génica eficaz en polluelo retinas para estudios de cultivo celular primario: Un<em&gt; Ex-ovo</em&gt; Enfoque electroporación

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

Investigación

Biología del Desarrollo

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 y Co-cultura de ratón ovarios y los folículos ováricos

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 electroporación y organotípicos rebanada Cultura de ratón Hipocampo 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.

Diferenciación neural de células madre embrionarias de ratón en el suero libre de monocapa 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.

Los explantes de retina enteras de pollo embriones para Tratamientos electroporación y Reactivos Químicos

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.

La transferencia de genes en el pollo auditiva de órgano de<em&gt; En Ovo</em&gt; Micro-electroporación

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 de adulto pez cebra transgénico de retina explantes de Células vivas imágenes de microscopía multifotónica

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.

Disección y cultivo de riñón embrionario de ratón

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.

Mediada por CRISPR pérdida de función análisis en gránulo cerebeloso células usando en el útero electroporación basado en la transferencia de genes

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.

Aislamiento y diferenciación de los mioblastos primarios de los explantes musculares esqueléticos de ratón

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