This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

NOTA: Todos los experimentos con animales se llevaron a cabo de acuerdo con la ley alemana y fueron aprobados por las oficinas gubernamentales en Tübingen. 1. Preparación de Micropipetas, Soluciones y Membranas <ol><li> Preparación de Micropipetas <ol><li> Tire micropipetas de vidrio con un extractor micropipeta con el siguiente programa: Heat: 540, Pull: 125, Velocidad: 20 y Delay: 140. Las cantidades longitud de la aguja a 5,5 cm. </li><li> Agujas de bisel utilizando un microgrinder para obtener un tamaño de la punta adecuada de 4 mm. Almacenar las agujas en una caja o 15 cm placa de Petri que se eviten daños de las puntas. </li></ol></li><li> Preparación de Soluciones <ol><li> Solución de ADN plasmídico <ol><li> Preparar ADN de plásmido que contiene el constructo de ADNc deseado utilizando un kit Maxi-prep libre de endotoxina de acuerdo con el protocolo del fabricante. </li><li> Ajuste solución de ADN plasmídico a una concentración final de 3 g / l (sin pico GFP vector) o 4 g /l (con 1 g de GFP pico vector) libre de endotoxinas en tampón Tris-EDTA que contiene verde rápido (concentración final 0,05%). </li></ol></li><li> Solución de laminina Stock <ol><li> Disolver 1 mg de laminina en agua estéril hasta un volumen final de 1 ml. Preparar alícuotas de 100 ly se almacena a -80 ° C. </li></ol></li><li> Poli-L-Lisina solución Stock <ol><li> Disolver 50 mg de poli-L-lisina en 50 ml de agua estéril a una concentración final de 1 mg / ml. Preparar alícuotas de 1 ml y se guardan en -20 ° C. </li></ol></li><li> Completa solución salina equilibrada de Hank (HBSS completo) <ol><li> Preparar completa HBSS mediante la combinación de 100 ml de HBSS 10x, 2,5 ml de 1 M de tampón HEPES (pH 7,4), 30 ml de 1 M D-glucosa, 10 ml de 100 mM CaCl 2, 10 ml de 100 mM MgSO 4, y 4 ml de 1 M de NaHCO3. Añadir agua estéril hasta 1 L y se almacena a 4 ° C. NOTA: Autoclave todas las soluciones de esperar tampón HEPES 1 M y 1 M D-glucosa, que son filter esterilizado. </li></ol></li><li> Rebanada medio de cultivo <ol><li> Preparar medio de cultivo rebanada mediante la adición de 35 ml de medio basal de Eagle, 12,9 ml de HBSS completo (1.2.4), 1,35 ml de 1 M D-glucosa, 250 l de 200 mM de L-glutamina, y 500 l de penicilina-estreptomicina a obtener un volumen final de 50 ml. Añadir suero de caballo a una concentración final de 5% y se almacena a 4 ° C. </li></ol></li><li> Punto de bajo punto de fusión (LMP) de agarosa <ol><li> Preparar una solución de agarosa LMP 4% mediante la adición de 2 g de agarosa LMP a 50 ml de HBSS completo (1.2.4), seguido por calentamiento en un horno de microondas durante 1-2 minutos a alta potencia. Mantener esta solución en un baño de agua a 37-39 ° C. Guarde la solución a 4 ° C y la reutilización. </li></ol></li><li> Solución de paraformaldehído <ol><li> En una campana de humos preparar una solución de 4% de paraformaldehído (PFA) mediante la adición de 4 g de paraformaldehído a 100 ml de PBS 1x. Calentar la solución a 60 ° C y añadir unas gotas de NaOH 1 N hasta que la solución se convierte en clear. </li></ol></li><li> Solución de permeabilización <ol><li> Disolver 9 g de BSA en 300 ml de 1x PBS que contenía 0,3% Triton X-100 y se almacena a 4 ° C. Añadir 10% de azida de sodio para almacenamiento a largo plazo. </li></ol></li></ol></li><li> Recubrimiento de insertos de membrana <ol><li> Diluir una alícuota de solución de stock laminina (1.2.2) y una parte alícuota de solución madre de poli-L-lisina (1.2.3) en agua estéril hasta un volumen final de 12 ml. </li><li> Coloque los insertos de membrana en placas de 6 pocillos conteniendo cada pocillo 2 ml de agua estéril. Añadir 1 ml de solución de recubrimiento en la parte superior de la membrana y se incuba O / N a 37 ° C en un incubador de CO2 5%. </li><li> Después de la incubación se lava la membrana inserta tres veces con 1 ml de agua estéril y seco. Use protectores de membrana recubiertos en el mismo día o se almacena a 4 ° C durante un máximo de cuatro semanas en una placa bien seco 6. </li></ol></li></ol> 2. Inyección de ADN y la electroporación de E15.5 y E18.5 embriones <ol><li&gt; Anestesie la vez acoplado hembra de ratón colocándolo en una cámara de anestesiante saturado con 5% de isoflurano y conectado a un vaporizador. Circular isoflurano y oxígeno a una velocidad de 1 L / min. Mantenga el animal en el cuadro de 2-4 minutos o hasta inconsciente que se prueba apretando entre las patas del ratón. </li><li> La eutanasia del ratón inconsciente por dislocación cervical en el día embrionario (E) 15.5 o 18.5. Diseccionar el útero contiene los embriones 13 y colocarlo en una placa de Petri que contiene 15 a 20 ml de HBSS frío completa. NOTA: A partir de este punto en adelante, mantener los embriones y tejidos en el hielo. </li><li> Use un par de tijeras para separar cada embrión a partir del cuerno uterino y el lugar en una segunda placa de Petri que contiene HBSS fría completa. </li><li> Bajo un microscopio de disección, cortar la pared muscular uterina y la placenta usando un par de pinzas finas (# 55) y tijeras. Suelte con cuidado el embrión desde el saco vitelino. </li><li> Use un par de tijeras de Bonn de decapitate los embriones justo por encima de las patas delanteras en un ángulo de 60 °. Si el experimento requiere el genotipado de los embriones, se recoge una muestra de tejido para el aislamiento de ADN genómico (un pequeño trozo de la cola). </li><li> Transferir la cabeza a un plato limpio y seco Petri. Debido a que la cabeza había sido decapitado en un ángulo de 60 °, la cabeza debe incline hacia un lado cuando se coloca dorsal hacia arriba. </li><li> Coloque una aguja cuidadosamente en el centro del hemisferio cerca de la bregma (Figura 1A, B). Inyectar aproximadamente 2-3 l (en 3 o 4 g / l) de solución de ADN pisando el pedal del Picospritzer III usando 30 libras de presión para la duración de 10-15 ms por impulso, aplicando 5-8 pulsos. La duración y el número de los impulsos depende del diámetro de la abertura de la aguja con aberturas más pequeñas que requieren más tiempo. El intervalo entre cada pulso asciende a 1 seg. </li><li> Antes de colocar los electrodos, aplique unas gotas de HBSS completo en la cabeza del embryo. Coloque los electrodos de tal manera que el terminal &quot;negativo &#39;está en el mismo lado que el ventrículo inyectado y el electrodo&quot; positiva &quot;en el lado opuesto del ventrículo inyectada debajo de la oreja de la cabeza del embrión (Figura 1C, D). Aplicar 5 pulsos de 50 V. <ol><li> Utilice 3 electrodos mm para E 15.5 y 5 mm para electrodos E 18.5. </li></ol></li></ol> 3. La disección del cerebro <ol><li> Después de la electroporación, retire la piel de la cabeza con la ayuda de un par de pinzas finas. El uso de un par de tijeras de primavera hacer una pequeña incisión en el medio del cerebelo en la línea media del cráneo. </li><li> Inserte las tijeras de primavera en la incisión y cortar longitudinalmente a lo largo de la sutura sagital. Despegar la calavera y separar el cerebro del cráneo mediante el uso de unas pinzas finas. Transfiera todo el cerebro en 15-20 solución HBSS completa ml frío. </li><li> Mientras tanto, se vierte 4% de agarosa LMP,se mantiene a 37-39 ° C en un baño de agua, en un molde de desprendimiento. </li><li> Tome el cerebro de la completa HBSS por una espátula pequeña pala y drenar el exceso de HBSS usando un pañuelo de papel fino o Kimwipes. </li><li> Coloque todo el cerebro suavemente en la agarosa y ajuste su posición con una aguja fina. Mantener el molde en hielo hasta que la agarosa se solidifica y el bloque se secciona (para secciones coronales los bulbos olfativos apuntan hacia arriba). </li></ol> 4. Vibratome Seccionamiento y rebanada Cultura <ol><li> Recorte los bloques de agarosa LMP y pegarlas en la platina utilizando &#39;pegamento&#39;. Después de que el pegamento se seca la fase de transferencia de la muestra a la bandeja de amortiguamiento de la vibratome y llene de hielo frío completar HBSS hasta que el bloque se encuentra inmersa en la solución. NOTA: Esterilizar todas las superficies de instrumentos y equipos con etanol al 70% antes de la sección. </li><li> Preparar 250 micras vibratome secciones gruesas utilizando una nueva hoja de la siguiente manera. <ol><li> Recorte el wi bloqueº los ​​siguientes ajustes; frecuencia - 60 Hz, amplitud - 0.7 micras, velocidad 16 a 18 mm / seg. Cortar las secciones que contienen el tejido deseado con los ajustes anteriores a velocidad lenta (9 mm / seg). Inicio del seccionamiento de la parte posterior del cerebro, recoger 5-7 secciones del cerebro. </li><li> Transferencia de las secciones a una placa de cultivo de 6 pocillos limpia que contiene 5 ml de HBSS enfriado con hielo completar con la ayuda de una espátula doblada y mantener en hielo hasta que todas las secciones se recogen (Figura 1E). </li></ol></li><li> Humedecer la membrana de una manera cruzada con 100 l de HBSS completo antes de colocar las secciones en la membrana, para facilitar la orientación de las secciones. </li><li> Transferir las secciones usando una espátula doblada sobre la membrana (recoger un rincón de la sección con unas pinzas y tire a la espátula y luego usar el fórceps para empujar la sección sobre la membrana). Coloque un máximo de cinco secciones en una membrana y organizar mediante el uso de fórceps (Figura 1F). No superponga las secciones entre sí. <ol><li> Tome el exceso de HBSS fuera de la membrana utilizando una pipeta. Las membranas específicas utilizadas aquí están unidos a un bastidor y se insertan en la placa de cultivo de tejido, lo que permite que el tejido esté en contacto pero no cubierto por el medio. </li></ol></li><li> Coloque los insertos de membrana en una placa de 6 pocillos que contiene 1,8 ml de medio de cultivo slice (1.2.5) (Figura 1G, H). Incubar la placa de cultivo a 37 ° C con 5% de CO2 durante 11 o 14 DIV DIV. Cambiar la mitad del medio (0,9 ml) cada segundo día. NOTA: En esta etapa, añadir los reactivos como bromodesoxiuridina (BrdU; 10 mM concentración final) para el etiquetado de la proliferación de células a los medios de comunicación por primera 20 horas del tiempo de cultivo. </li></ol> 5. Fijación de las Secciones Seguido por inmunofluorescencia tinción <ol><li> Utilice una hoja de bisturí limpio y afilado para cortar y recortar las membranas, dependiendo de la orientación delas secciones. </li><li> Transferencia de las secciones, junto con la membrana a una placa de 24 pocillos que contenía 1 ml de PFA al 4% (1.2.7). Incubar las secciones durante 1 hora a RT seguido de 3 lavados con PBS 1x durante 15 min cada uno. Incubar las secciones O / N con solución de permeabilización a 4 ° C con agitación suave. </li><li> El día siguiente, se incuban las secciones con anticuerpos primarios apropiados, diluidos en solución de permeabilización, O / N o durante 48 horas a 4 ° C con agitación suave. </li><li> Lávese las secciones 3 veces durante 15 minutos con PBS 1x y se incuba O / N a 4 ° C con los anticuerpos secundarios apropiados diluido en solución de permeabilización. </li><li> Después de la incubación con anticuerpos secundarios, lavar las secciones una vez con 1x PBS durante 15 min seguido de tinción con DAPI durante 10 min. </li><li> Lávese las secciones 3 veces con 1x PBS durante 15 minutos cada uno y traslado al microscopio. Añadir ImmunoMount y colocar suavemente un cubreobjetos en la parte superior de las secciones. Seque el portaobjetos O / N a 4 ° C y seal con esmalte de uñas. NOTA: Mantenga siempre las diapositivas a 4 ° C. <ol><li> Analizar los cultivos de corte por microscopía confocal (Figura 1I). </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

El hipocampo tiene una función importante en el aprendizaje y la memoria. El giro dentado es también una de las dos regiones del cerebro donde se produce la neurogénesis no sólo durante el desarrollo sino también durante la edad adulta. Postnatal y adulto neurogénesis del hipocampo procede de manera similar que implica muchos factores comunes. La definición de los mecanismos de regulación de estos factores va a ser muy útil en la comprensión de las enfermedades neurodegenerativas que a su vez dará lugar a nue...

El hipocampo desempeña un papel importante en la memoria y el aprendizaje, así como el comportamiento emocional. Una función principal consiste en la consolidación de la memoria a corto plazo en memoria a largo plazo, lo que requiere una alta plasticidad del sistema nervioso. El giro dentado del hipocampo actúa como puerta de entrada principal de información de entrada y es también una de las dos regiones del cerebro con la neurogénesis continua a lo largo de la edad adulta 1,2. El desarrollo de la estructura del hipocampo se produce durante la embriogénesis tardía y en particular durante los primeros 3 a 4 semanas después del parto 3. Durante el desarrollo temprano de la circunvolución dentada de un grupo de células madre está establecido requiere para postnatal, así como la neurogénesis adulta 4. Las neuronas en desarrollo pasan a través de diversas etapas, desde la célula madre a través de varias etapas de células progenitoras a los inmaduros y, finalmente, la neurona madura durante postnatal así como la neurogénesis adulta. En diferentes etapas de la neurogénesis la expresión degenes específicos se requiere para permitir la maduración y la integración de nuevas neuronas en los circuitos del hipocampo 5,6. Usando la genética del ratón y análisis de fenotipo por inmunohistoquímica así como los métodos moleculares permitió definir el patrón de expresión y la función de muchos de estos genes. Además el análisis de microarrays, así como la cromatina immunoprecipitation (CHIP) proporcionado información sobre posibles genes diana directos e indirectos 7,8. Sin embargo, todavía hay muchas preguntas abiertas acerca de los mecanismos de regulación del desarrollo del hipocampo, en particular el desarrollo del giro dentado. Para obtener una mayor comprensión de cómo los genes específicos se regulan un sistema es necesario permitir la manipulación de un pequeño número de células por abajo o sobre regulación del gen de interés y / o de sus genes diana y seguir su destino durante el desarrollo. En electroporación in utero de shRNAs, cDNA de genes de interés o Cre recombinase proporciona una herramienta de este tipo. Para asegurar la presencia del ADN deseado o ARNs pequeños plásmidos de expresión se debe utilizar para la electroporación. Este enfoque se implementa con mucho éxito en el estudio del desarrollo cortical 9,10, pero es un enfoque más difícil examinar el desarrollo de la circunvolución dentada debido a la posición de las estructuras del hipocampo en las capas más profundas del cerebro. Electroporación utero Ex seguido por cultivo de cortes organotípicos es un enfoque para evitar este problema 11,12. En contraste con la electroporación en el útero no todo el embrión, pero sólo la cabeza se utiliza permitiendo por lo tanto para colocar los electrodos de una manera más favorable para dirigir el shRNA / ADN hacia el hipocampo y la circunvolución dentada. Nuestro grupo emplea con éxito fuera del útero de la electroporación para estudiar el papel del factor de transcripción Bcl11b durante el desarrollo del giro dentado 8. Bcl11b tiene un doble papel en el desarrollo giro dentado por regulating proliferación de células progenitoras, así como la diferenciación como quedó demostrado por inmunohistoquímica. Para definir además un mecanismo para la participación Bcl11b en estos procesos, protocolos del grupo Polleux 11,12 se ajustaron para estudiar la circunvolución dentada como se describe a continuación en la sección de protocolo. En una primera aproximación a la cuestión se abordó si Bcl11b está regulando la diferenciación celular de las células neuronales de forma autónoma. Un segundo enfoque examinó si Desmoplakin, un gen diana directa de Bcl11b, es suficiente para rescatar el fenotipo Bcl11b.

<table border="0" cellpadding="0" cellspacing="0" width="271">
	<tbody>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Name of Reagent/ Equipment</td>
			<td style="width:73px;">Company</td>
			<td style="width:63px;">Catalog Number</td>
			<td style="width:64px;">Comments/ Description</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Flaming/ Brown Micropipette Puller</td>
			<td style="width:73px;">Sutter Instruments Company (USA)</td>
			<td style="width:63px;">P-97</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fine Glass Pipettes</td>
			<td style="width:73px;">Warner Instruments</td>
			<td style="width:63px;">G100F-4</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:71px;">Microgrinder</td>
			<td style="width:73px;">Narishige, Japan</td>
			<td style="width:63px;">EG-44</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:71px;">Anesthetic Bracket unit</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0412</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Halovet Vaporizer</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0398</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fluovac System</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0387</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">IMS Fluosorber</td>
			<td style="width:73px;">Harvard Apparatus</td>
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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.

La ablación del factor de transcripción Bcl11b hace que el deterioro de la proliferación de células progenitoras y la diferenciación neuronal que resulta en un tamaño giro dentado reducida y número de células. Además neuronas mutantes no pueden integrarse en los circuitos del hipocampo causando el aprendizaje y el deterioro de la memoria 8. Para responder a preguntas sobre el mecanismo regulador (s) de Bcl11b en estos procesos se empleó electroporación ex utero. <p class="jove_content"...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

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 ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

9.4K vistas.  University of Ulm. 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.

Video: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

La inducción de la proteína a través de Supresión<em&gt; In Utero</em&gt; La electroporación para definir Déficits en Neuronal migración en modelos transgénicos

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Investigación

Biología del Desarrollo

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 ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

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

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 ...

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Culturas organotípicos rebanada de Estudios de Postnatal neurogénesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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

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

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

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

Ex Vivo Cultura de polluelo cerebelosos Rebanadas y espacialmente focalizadas Electroporación de gránulos precursores celulares

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

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Disección y preparación del segmento Coronal de desarrollo de hipófisis de ratón

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

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 ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Cultura Organotypic método para estudiar el desarrollo de tejidos de embrión de pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.