Name: whole retinal explants from chicken embryos for electroporation and chemical reagent treatments
Uploaded: 2015-09-30T15:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments at JoVE.com

The work was supported by Barncancerfonden (PR2013-0104), Swedish Research Council (12187-18-3), &#246;gonfonden, Kronprinsessan Margaretas arbetsn&#228;mnd f&#246;r synskadade, Synfr&#228;mjandets forskningsfond and St Eriks &#246;gonsjukhus forskningsstipendier.

Este protocolo se realiza de acuerdo con las recomendaciones de la &quot;Guía para el Cuidado y Uso de Animales de Laboratorio de la Asociación para la Investigación en Visión y Oftalmología&quot;. 1. Manipulación del huevo y la recogida de los ojos <ol><li> Almacene los huevos Leghorn blancas a 12-14 ° C durante no más de 1 semana. Si está disponible, utilice un enfriador de vino refrigerada para almacenar los huevos fertilizados. Nota: Las temperaturas más altas del almacén conducen a un desarrollo anormal de los embriones, mientras que las temperaturas más bajas aumenta la mortalidad. Tiempo de almacenamiento extendido aumenta tanto la mortalidad y el desarrollo anormal. </li><li> Incubar los huevos en un 37,5 ° C y 60% humidificado incubadora de huevos con suave balanceo a una frecuencia de 5 veces por 24 horas. </li><li> Retire los huevos incubados de la incubadora de huevos a la edad embrionaria deseado. Nota: La edad del embrión de pollo se determina como el tiempo de incubación y se denota como día embrionario (E) o como etapas (st), De acuerdo con la serie de etapas de desarrollo descritos por Hamburguesa y Hamilton 12. </li><li> Coloque los huevos seleccionados en la campana de flujo laminar. Limpiar la cáscara del huevo con etanol al 70% para evitar la contaminación del embrión. Nota: Llevar a cabo todos los procedimientos en condiciones asépticas con soluciones estériles <url> http://www.jove.com/science-education/5030/making-solutions-in-the-laboratory&quot;lutions </url> e instrumentos. </li><li> Toque en el lado del huevo firmemente contra una superficie dura para abrir una grieta en el huevo. Coloque el contenido en un 100 mm placa de Petri. Use una pequeña cuchara para transferir el embrión a una placa de Petri de 35 mm que contiene precalentado (37 ° C) 1x PBS para evitar que el tejido de secado. </li><li> Coloque la placa de Petri de 35 mm que contiene el tejido embrionario en un microscopio de disección estereoscópica binocular en la campana de flujo laminar. Decapitar el embrión apretando el cuello con unas pinzas finas y descartar el cuerpo. </li><li> Utilice unas pinzas finas para hacer una incisión ªáspera la boca, ventral a la vista. Comenzando en el sitio de la incisión, rasgar el tejido que rodea todo el ojo. Pellizcar el nervio óptico y quitar el ojo intacto de la cuenca del ojo. Recoge tanto el ojo izquierdo y derecho desde el mismo embrión para su uso, ya sea como el control o el ojo tratado. </li><li> Use una cuchara pequeña para transferir los ojos a una nueva placa de Petri de 35 mm que contiene precalentado (37 ° C) 1x PBS. </li></ol> 2. Preparación de toda la retina explantes <ol><li> Utilice unas pinzas finas para retirar todo el tejido alrededor del ojo incluyendo la anlage escleral. Nota: El tejido se separará fácilmente dejando la retina en el cuerpo vítreo y la lente con el epitelio de pigmento intacta (Figura 1A-B). </li><li> Utilice unas pinzas finas pellizcar una pequeña incisión en el epitelio pigmentario en la parte dorsal del ojo sin dañar la retina neural (Figura 1C). </li><li> Utilice unas pinzas finas para arrancar suavemente abra la starti epitelio pigmentariong en el sitio de incisión, dejando la lente y toda la retina unido al cuerpo vítreo (Figura 1D). Mantener el ojo en caliente (37 ° C) 1x PBS para facilitar la eliminación del epitelio pigmentario. Nota: El epitelio de pigmento puede ser difícil de eliminar, en particular, a lo largo del cuerpo ciliar alrededor de la pupila, en la región anterior del ojo, ya lo largo de la fisura coroides. Por lo tanto, algunos de epitelio pigmentario se puede dejar en la retina neural (Figura 1E-F). </li></ol> 3. El tratamiento de toda la retina explantes <ol start="1"><li> Electroporación de explantes de retina enteros Nota: La electroporación de los explantes de retina enteros puede realizarse directamente después de la etapa 2. <ol><li> Preparar medio de cultivo que contiene 1: 1 DMEM: F12 mezcla de nutrientes, 10% de FBS, 10 U / ml de penicilina estreptomicina, 5 mg / ml de insulina, y 2 mM L-glutamina. </li><li> Corte un 1,5 ml desechables cubeta de plástico (12,5 mm x 12,5 mm x 45 mm) (o cualquier pequeño recipiente capaz de contener la solución de 300-400 l) aproximadamente 8 mm de la parte inferior usando una sierra de hoja fina. </li><li> Diluir el plásmido de ADN de elección en 1xDPBS a una concentración final de 0,1 mg / l. </li><li> Encienda los parámetros electroporador y set de 15 V para ST20 - ST25 (E3-E4) retina y 20 V para ST26 - ST27 (E5) retina. Ajuste los pulsos repeticiones de 5 pulsos de 50 ms de pulso de longitud con intervalos de 1 seg. Utilice un generador de impulsos que puede ser controlado por un pedal en el suelo como se necesitarán ambas manos para sostener los electrodos en su lugar. </li><li> Conecte dos paleta en forma de (, diam circular plana. 4 mm) electrodos de platino (Figura 2A) a la toma de salida del generador de impulsos. Nota: Los electrodos de platino utilizados en este protocolo fueron hechas por el taller de la universidad. Los electrodos fueron hechas de placa de platino de 0,1 mm &quot;rondelles&quot; (grado de platino: 950 Pt / 5 Cu). La conexión de 0,8 mm wire se aisló utilizando un tubo de plástico. </li><li> Transferir cuidadosamente todo el explante de la retina desde el paso 2.3 a la cubeta (o recipiente pequeño equivalente) usando una pipeta Pasteur de polietileno con la punta cortada para adquirir el tamaño adecuado punta de apertura para el explante de la retina. Evite el uso de puntas de pipeta como la retina tiende a unir a la de plástico y desgarrar. </li><li> Utilice una pipeta 100 l para eliminar suavemente todo el 1x PBS que rodea el explante teniendo cuidado de no tocar la retina la retina para evitar que se rompa. </li><li> Añadir solución de plásmido 100 l a la cubeta para cubrir todo el explante de la retina. Presione suavemente el explante de la retina con pinzas si flota a la cima. El uso de fórceps o electrodos de situar el objetivo hacer frente a cualquier un lado de la cubeta y el nervio óptico hasta el fondo de la cubeta. </li><li> Coloque el electrodo positivo en frente de la lente y el electrodo negativo detrás de la retina (Figura 2A), sin tocar el tejido. </li><li>Aplique la corriente presionando el pedal. Compruebe si hay burbujas en los electrodos que indican la descarga con éxito. </li><li> Usar una pipeta de 100 l para eliminar toda la mezcla de plásmido a partir de la cubeta sin dañar la retina. La mezcla de plásmido puede ser reutilizado tres a cinco veces. </li><li> Llenar la cubeta con precalentado (37 ° C) 1x PBS. El uso de fórceps para desprender suavemente el explante de la retina de la pared de la cubeta. Después de la electroporación la retina a veces se cubrió con burbujas, lo que hace que se adhiera a la pared de la cubeta. </li><li> Utilice la pipeta Pasteur de polietileno para transferir todo el explante de la retina en una placa de 24 pocillos que contenía 1 ml precalentado (37 ° C) medio de cultivo por pocillo. Incubar un explante de la retina por pocillo. </li><li> Incubar el explante de la retina a 37 ° C en un agitador rotatorio, con una velocidad constante de 50 rpm, dentro de un incubador de células con 5% de CO 2 durante 24 horas. La rotación es asegurar la máxima exposición al medio y para prevent adherencia de la retina a la parte inferior de la placa de 24 pocillos. </li><li> Continúe con el paso 4. </li></ol></li><li> El tratamiento de los explantes de retina enteras con reactivos químicos Nota: El tratamiento químico de los explantes de retina enteros puede realizarse directamente después de la etapa 2. <ol><li> Preparar medio de cultivo que contiene 1: 1 DMEM: F12 mezcla de nutrientes, 10% de FBS, 10 U / ml de penicilina estreptomicina, 5 mg / ml de insulina, y 2 mM L-glutamina. </li><li> Use una cuchara pequeña para transferir el explante de la retina desde el paso 2.3 en una placa de 24 pocillos que contenía 1 ml precalentado (37 ° C) medio de cultivo por pocillo. Incubar un explante de la retina por pocillo. </li><li> Incubar toda la retina explante durante 60 min a 37 ° C en un agitador rotatorio, con una velocidad constante de 50 rpm, dentro de un incubador de células con 5% de CO 2 antes de añadir un reactivo químico o vehículo. </li><li> Preparar la solución química, por ejemplo Cdk1 / 2 inhibidor III, un inhibidor de la progresión de la fase M-13, en una finalconcentración de 30 mM en DMSO. </li><li> Retire la placa de 24 pocillos, que contiene todo el explante de la retina, de la incubadora celular. Añadir 10 l de reactivo químico o el vehículo (DMSO) directamente al medio de la retina, resultando en una concentración final de 300 nM Cdk1 / 2 inhibidor III en 0,01% de DMSO. Girar manualmente la placa de 24 pocillos para permitir una distribución uniforme del reactivo químico o vehículo en la solución. </li><li> Incubar los explantes de retina a 37 ° C en un agitador rotatorio, con una velocidad constante de 50 rpm, dentro de un incubador con 5% de CO 2 durante el tiempo deseado (hasta 24 h). </li><li> Continúe con el paso 4. </li></ol></li></ol> 4. Fijación y Congelación de Whole Retina Eexplants <ol><li> Retire la placa de 24 pocillos que contiene el explante de la retina entera tratada de la incubadora celular. </li><li> Use una cuchara pequeña para transferir el explante de la retina a una placa de 24 pocillos que contienen 1 ml / pocillo helada paraformaldehído al 4% en 1xPBS. Incubate en hielo durante 15 min. </li><li> Use una cuchara pequeña para transferir todo el explante de la retina a una placa de 24 pocillos que contienen 1 ml / pocillo 1xPBS frío de hielo para lavar la retina. Incubar en hielo durante 10 min. </li><li> Use una cuchara pequeña para transferir todo el explante de la retina a una placa de 24 pocillos que contienen 1 ml / pocillo frío hielo sacarosa 30% en 1xPBS. Incubar en hielo durante 1-3 hr dependiendo del día embrionario de la retina. Para ST20 - ST25 (E3) retinas, se incuban durante 1 hora, retinas mayores necesitan más tiempo de incubación en sacarosa. </li><li> Use una cuchara pequeña para transferir todo el explante de la retina sobre una película de parafina con un contenido medio de montaje de aproximadamente 500 l de congelación criostato. Retire la mayor cantidad de sacarosa posible moviendo suavemente el explante de la retina en el medio de congelación utilizando la cuchara pequeña. </li><li> Transferir el explante de la retina a un molde de inclusión que contiene medio de congelación. Coloque el ojo para facilitar la futura seccionamiento. Ponga el molde de inclusión que contiene todo el explante de la retina en dry hielo hasta que el medio se congela sólido. Almacenar a -80 ° C. </li></ol>

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The authors declare that they have no competing financial interests.

En este trabajo se presenta un protocolo detallado para la disección, la electroporación o tratamiento químico, y el cultivo de los explantes enteros retina de embriones de pollo. Este protocolo es fácil, rápido y permite tanto una alta tasa de éxito y reproducible resultados. Electroporación de explantes de retina enteros produce grandes áreas de células que expresan el constructo de gen de interés. Es fácil de colocar correctamente los electrodos y para expone...

La retina es parte del sistema nervioso central y es, con su relativa simplicidad y la arquitectura celular bien caracterizada, un modelo popular para estudiar el desarrollo del sistema nervioso central. El ojo del embrión de pollo es relativamente grande en comparación con el resto del embrión. Por tanto, es de fácil acceso en ovo de manipulaciones experimentales, tales como inyecciones o electroporación, y sirve como una excelente herramienta para adquirir conocimientos sobre las células de la retina y la biología del desarrollo in vivo. A pesar de estas ventajas importantes, la supervivencia de los embriones puede ser baja cuando los experimentos son invasivas como con electroporaciones, inyecciones repetidas, o manipulaciones experimentales combinados. La electroporación de plásmidos de ADN en el embrión de pollo in ovo es un importante y bien establecida técnica 1. Permite para el etiquetado de las neuronas, la localización del destino celular, así como tractos neuronales en el centro de nervous sistema y permite ectópico expresión génica para analizar la función de proteínas in vivo. La técnica ha sido utilizada para los estudios de tubo neural 2, hindbrain 3, 4 y la retina. Electroporación de retina embrionaria in ovo tiene algunas dificultades experimentales que están relacionados con la situación in vivo. La posición del ojo, debido a la plegado craneal del embrión, es relativamente cerca del corazón. Esta proximidad aumenta el riesgo de un paro cardíaco después de la electroporación, y el riesgo aumenta con la edad del embrión. Por otra parte, para acceder al ojo, es necesario abrir las membranas embrionarias, aumentando así el riesgo de sangrado, malformaciones y posterior viabilidad reducida. Al probar y optimizar un nuevo plásmido de ADN a menudo sin un resultado fenotípico conocido, estas limitaciones pueden disminuir la eficacia y el poder del método incluso para un experimentalista experimentado. Tal como se presenta en este protocolo, la cultura de la wexplante agujero retinal, que se define como a toda la retina neural con el epitelio de pigmento eliminado, es un método eficaz que complementa el enfoque en ovo. Inyecciones intraoculares de reactivos químicos son relativamente fáciles de realizar in ovo. Sin embargo, la concentración eficaz y exacta de los reactivos inyectados dentro de la retina neural puede ser difícil de controlar totalmente. El volumen inyectado puede variar debido a las fugas y el sitio exacto de la inyección puede afectar tanto a la distribución del reactivo dentro del ojo y de la difusión a través del cuerpo vítreo. La variabilidad tendrá importantes implicaciones para la interpretación de los resultados cuando es decir, se determina una curva dosis-respuesta para un inhibidor de la enzima; particularmente si el efecto es pequeño y la ventana temporal del efecto es estrecho. Por otra parte, un solo ojo puede ser utilizado de cada embrión cuando se realiza en experimentos in ovo debido a los posibles efectos sistémicos a través del torrente sanguíneosobre el ojo contralateral. Coincidente edad es importante a la hora de estudiar el desarrollo y la variabilidad individual entre tratados y los embriones de control pueden conducir a la variabilidad experimental adicional. Por estas razones, un ex ovo método basado en explantes de retina de embriones de pollo fue desarrollado, en el que la retina neural puede ser expuesto a un uniforme y controlada condición experimental in vitro. El presente protocolo fue desarrollado en base a protocolos anteriores 5-9. Explantes de retina de la etapa (st) 20 (días embrionarias [E] 3) a ST31 (E7) embriones de pollo fueron disecados, culta y electroporación con una concentración de ADN plásmido definido o expuestos a un medio que contiene una concentración definida de un reactivo químico. El protocolo que aquí se presenta ha sido implementado con éxito en publicaciones recientes, utilizando varios reactivos químicos diferentes, incluyendo los reguladores de la vía de daño en el ADN, como KU55933, SB 218078 y NSC 109555 ditosylate, y el ciclo celular, tal como Cdk1 / 2 inhibidor III 10,11.

<table><tbody><tr><td>1xPBS (tablet)</td><td>Life technologies</td><td>18912-014</td><td></td></tr><tr><td>10x DPBS</td><td>Life technologies</td><td>14080-048</td><td></td></tr><tr><td>100 mm Petri dish</td><td>VWR</td><td>734-0006</td><td></td></tr><tr><td>100 &amp;mu;l pipette tips</td><td>VWR</td><td>613-0798</td><td></td></tr><tr><td>1.5 ml disposable plastic cuvette</td><td>Thomas Scientific</td><td>8495V01</td><td></td></tr><tr><td>24-well plate</td><td>Sigma Aldrich</td><td>D7039</td><td></td></tr><tr><td>35 mm Petri dish</td><td>VWR</td><td>391-1998</td><td></td></tr><tr><td>70% ethanol</td><td>Solveco</td><td>1054</td><td></td></tr><tr><td>Cdk1/2 inhibitor III</td><td>217714</td><td>Calbiochem</td><td>300 nM in 0.01% DMSO</td></tr><tr><td>Cell culture incubator</td><td>Thermo Forma</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>41966-029</td><td></td></tr><tr><td>Electrodes</td><td></td><td></td><td>Platina, custom made</td></tr><tr><td>Electro square porator ECM 830</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>F12 Nutrient mix</td><td>Life Technologies</td><td>31331-028</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Forceps</td><td>AgnThos</td><td>0108-5-PS</td><td></td></tr><tr><td>Freezing medium NEG50</td><td>Cellab, Sweden</td><td>6502</td><td></td></tr><tr><td>GFP expressing DNA plasmid (pZGs)</td><td></td><td></td><td></td></tr><tr><td>Humidified incubator</td><td>Grumbach Brutgeraete GmbH, Asslar, Germany</td><td>8204</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I9278-5ML</td><td></td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030024</td><td></td></tr><tr><td>Mounting medium ProLong Gold with DAPI</td><td>Life Technologies</td><td>P36935</td><td></td></tr><tr><td>Paraffin film</td><td>VWR</td><td>291-1214P</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>16005-1KG-R</td><td></td></tr><tr><td>Peel-A-Way embedding mold&amp;nbsp;</td><td>Sigma Aldrich</td><td>E6032</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>PhosphoHistone 3 (PH3)&amp;nbsp;</td><td>Millipore</td><td>06-570</td><td>Dilution 1/4,000</td></tr><tr><td>Platinum electrodes (custom made from &quot;rondelles&quot;)</td><td>Sargenta</td><td>390-R (rondeller)</td><td>Dia: 4mm, 0.1 mm thickness</td></tr><tr><td>Platimun electrodes&amp;nbsp;</td><td>Sonidel</td><td>CUY700P4L</td><td>Dia: 4 mm</td></tr><tr><td>Polyethylene pasteur pipette</td><td>VWR</td><td>612-2853</td><td></td></tr><tr><td>Rotator shaker</td><td>VWR</td><td>444-2900</td><td></td></tr><tr><td>Small spoon</td><td>VWR</td><td>231-2151</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>443815S</td><td></td></tr><tr><td>White Leghorn eggs</td><td></td><td></td><td>Local supplier</td></tr><tr><td>Wine cooler</td><td>WineMaster 24, Caso, Berlin Germany</td><td></td><td></td></tr></tbody></table>

whole retinal explants from chicken embryos for electroporation and chemical reagent treatments

La retina es un buen modelo para el sistema nervioso central en desarrollo. El gran tamaño del ojo y lo más importante la accesibilidad para las manipulaciones experimentales en ovo / in vivo hace que el pollo retina embrionaria un modelo experimental versátil y muy eficiente. Aunque la retina de pollo es fácil dirigirse in ovo mediante inyecciones intraoculares o la electroporación, la concentración eficaz y exacta de los reactivos dentro de la retina puede ser difícil de controlar completamente. Esto puede ser debido a las variaciones de la zona de inyección exacta, la fuga desde el ojo o desigual difusión de las sustancias. Además, la frecuencia de malformaciones y mortalidad después de manipulaciones invasivos tales como la electroporación es bastante alta. Este protocolo describe un ex ovo técnica para el cultivo de explantes de retina enteros a partir de embriones de pollo y proporciona un método para la exposición controlada de la retina a los reactivos. El protocol describe cómo diseccionar, experimentalmente manipular y explantes de cultivo enteros de la retina a partir de embriones de pollo. Los explantes pueden cultivarse durante aproximadamente 24 h, y ser sometidos a diferentes manipulaciones tales como la electroporación. Las principales ventajas son que el experimento no es dependiente de la supervivencia del embrión y que la concentración del reactivo introducido se puede variar y controlado con el fin de determinar y optimizar la concentración efectiva. Además, la técnica es rápida, barata y junto con su alta tasa de éxito experimental, se asegura reproducible resultados. Cabe destacar que sirve como un excelente complemento para los experimentos realizados in ovo.

Este protocolo describe la preparación (Figura 1A-F) y el cultivo de los explantes de retina enteros a partir de embriones de pollo. Este protocolo se ha usado con éxito para explantes de retina a partir de embriones enteros de ST20 (E3) a ST31 (E7). La electroporación de plásmidos de ADN en explantes de retina enteros permite para el etiquetado y el rastreo de las células progenitoras de la retina o sobre-expresión de diferentes productos génicos. Para los experiment...

Watch this Scientific Journal Video about Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments at JoVE.com

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

Este protocolo describe un método para diseccionar, experimentalmente manipular y explantes de cultivo completo de la retina a partir de embriones de pollo. Los cultivos de explantes son útiles cuando alta tasa de éxito, la eficacia y reproducibilidad son necesarios para probar los efectos de plásmidos para la electroporación y / o sustancias de reactivos, es decir, inhibidores enzimáticos.

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 ...

whole-retinal-explants-from-chicken-embryos-for-electroporation

Research

JoVE Journal

Developmental Biology

9.4K vistas.  Uppsala University.  Este protocolo describe un método para diseccionar, experimentalmente manipular y explantes de cultivo completo de la retina a partir de embriones de pollo. Los cultivos de explantes son útiles cuando alta tasa de éxito, la eficacia y reproducibilidad son necesarios para probar los efectos de plásmidos para la electroporación y / o sustancias de reactivos, es decir, inhibidores enzimáticos. 

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

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

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

En Electroporations Ovo de embriones de pollo HH Etapa 10

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biología

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Neurociencias

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

En electroporación in ovo en el mesencéfalo polluelo para estudiar la función génica en el desarrollo neuronal dopaminérgica

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

La electroporación in vivo en el útero y el ex de la expresión génica en células de ratón ganglionares retinianas

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Visualización de la migración celular tangencial en el Tectum óptico de Chick en desarrollo

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

En electroporación in ovo en la retina de embriones de pollo

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferencia de genes en embriones de pollo por los mayores<em&gt; Ex ovo</em&gt; La electroporación

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Cultivos Enriquecidos con Conos De La Retina De Embriones De Pollo Para Estudiar Las Interacciones Celulares De Varilla A Cono

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

Enfoque de pérdida de función en la retina embrionaria de pollo mediante el uso de la expresión transgénica mediada por transposones Tol2 de microARN artificiales

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

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

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