We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

Todos los procedimientos descritos en este trabajo se realizaron de acuerdo a las pautas recomendadas por el Cuidado de Animales y el empleo Comisión de la Universidad Johns Hopkins. 1. Antes de Tiempo: Preparación de Instrumentos, reactivos y Platos <ol><li> Preparación de los huevos: Incubar huevos de gallina fertilizados White Leghorn a 37,5 ° C y una humedad relativa del 60% durante 5 días en una incubadora de huevos. NOTA: Una incubadora con balanceo de la capacidad puede ser útil para la normalización y la sincronización de desarrollo embrionario, pero oscilante no es estrictamente necesario para el resultado de este protocolo. </li><li> Preparación de plásmido: <ol><li> Diseño plásmido constructo para la sobreexpresión de genes o regulación a la baja (por ejemplo, mediante el uso de la tecnología del RNAi 1,34) según sea necesario. Incluya un gen indicador (por ejemplo, EGFP, RFP o LacZ) en la construcción siempre que sea posible. NOTA: Algunos promotores de uso común, tales como el CMV, pueden llegar a ser silenciada en el tiempo. El promot CAGer (pollo beta-actina promotor con promotor de CMV) es una muy buena alternativa, proporcionando eficiente expresión a largo plazo del gen de interés 35-37. </li><li> Preparar una solución de plásmido a una concentración de 1,5 g / l en PBS estéril. OPCIONAL: Preparar la solución plásmido antes de tiempo y almacenarlo congelado hasta que esté listo para su uso. NOTA: Superior concentraciones de plásmido no dan lugar a un aumento significativo en la eficiencia, mientras que el uso de concentraciones más bajas de plásmidos disminuyen la eficiencia de transfección 1. </li></ol></li><li> Electrodos: <ol><li> Para el uso de ánodos de oro gruesa punta del electrodo (véase la Tabla de Materiales Específicos / Equipo). Limpie con etanol al 70%. NOTA: Aislamiento del oro punta de electrodo, dejando aproximadamente 0,5 mm expuestos, es aconsejable minimizar la fuga de la corriente eléctrica. Información para material aislante se puede encontrar en la Tabla de materiales específicos. </li><li> Hacer un cátodo de un cuadrado de 2,5 mm bfilamento de buey, de 4,5 mm de ancho (normalmente utilizado en extractores micropipeta). Con la ayuda de unas pinzas, deshacer la caja cuadrada y enderezar filamento en una tira larga. Luego, bajo microscopio de disección y usando una regla como guía, doblar el filamento en una forma cuadrada &#39;U&#39; 3 mm de largo en la base y 2 mm de alto en un lado, con el otro lado de la longitud restante del filamento (Figura 1 , C - D). Con unas pinzas, sumerja el electrodo en etanol al 96% y la llama para esterilizar. A continuación, conecte un cable eléctrico con una pequeña pinza de conexión para el brazo del electrodo más largo. </li></ol></li><li> Cámara electroporación de configuración: <ol><li> Preparar la cámara de electroporación mediante la reducción de la tapa de un tubo de microcentrífuga de 1,5 ml estéril y la colocación de ella, lado plano hacia abajo, a una placa de Petri de 100 mm usando cinta (Figura 1E). NOTA: Otros recipiente en forma de un pozo de tamaño similar funcionaría; sin embargo, un tubo de microcentrífuga de tapa es fácilmente accesible y autoclavable. El biendebe ser lo suficientemente para alojar un electrodo de ojo de pollo y el cátodo grande, pero no tan grande como para resultar en una pérdida de solución de plásmido. </li></ol></li></ol> 2. Ex Procedimiento electroporación in ovo <ol><li> Preparación de los instrumentos: <ol><li> Esterilizar las herramientas de micro-disección (2 pares de grandes pinzas Moloney curvos para la apertura de las cáscaras de huevo y sacando embriones, 2 pares de punta fina curvas pinzas Dumont para la eliminación de RPE, un par de Bonn dentado pinzas para quitar la lente y vítreo) por inmersión de la consejos en etanol al 96% y en llamas. Alcance la disección Limpie con trapos mojados en etanol al 70%. Calentar una botella de solución salina equilibrada estéril calcio-magnesio-libre de Hank (CMF-HBSS) en un baño de agua a 37 ° C. Rellene 100 mm placas de Petri estériles a 3/4 de su capacidad con una cálida CMF-HBSS. </li><li> Coloque la cámara de electroporación bajo otro microscopio de disección limpia y llena con 120 l de solución de plásmido. Conectar los electrodos al aparato de electroporación, haciendoSeguro electrodo de medida está conectado al polo negativo y el oro con punta de electrodo al polo positivo. </li></ol></li><li> Embrión Colección: <ol><li> Limpie los huevos limpiando conchas con toallitas húmedas en etanol al 70%. Mantener procedimiento estéril durante todo el resto del protocolo para evitar llevar la contaminación en los cultivos. El uso de grandes pinzas Moloney curvas abrir cuidadosamente un agujero en la cáscara del huevo golpeando suavemente en la punta redondeada de un huevo (más de la cámara de aire) y luego agarre y sacando trozos de concha. </li><li> Con otro par de pinzas quitar las membranas y recoger embriones por sacar con pala fuera del huevo (con cuidado de no dañar el embrión). Colóquelo en un plato con CMF-HBSS. NOTA: Recoger la mayor cantidad embriones como sea necesario para obtener el número deseado de vasos de la retina para la electroporación como se especifica en 2.4.4 </li><li> PASO CRÍTICO: Etapa de los embriones de acuerdo a Hamburger y Hamilton 38. NOTA: Para obtener embriones óptimos de eficienciadebe ser en la etapa 27 (Figura 2A). </li><li> La eutanasia a los embriones por decapitación utilizando fórceps Moloney. </li></ol></li><li> La obtención de Copas de la retina: <ol><li> Usando pinzas Dumont finas, enuclear cuidado de los ojos y la transferencia a un nuevo plato CMF-HBSS. </li><li> A partir de la parte posterior de cada ojo, cerca de la cabeza del nervio óptico y utilizando dos pinzas Dumont finas, introducir un consejo de cada par de pinzas entre la retina neural y RPE y cuidadosamente diseccionar el EPR ser cauteloso para no dañar el neuronal retina (Figura 2B). NOTA: La falta de Ca 2+ y Mg 2+ en la solución ayudará a la RPE desprenda de la retina neural más fácilmente. </li></ol></li><li> Electroporación: <ol><li> Establecer parámetros de electroporador para entregar 5 pulsos cuadrados de 15 voltios, 50 mseg de duración y 950 intervalo de milisegundos. Place &#39;U&#39; en forma de electrodo negativo dentro de la cámara de electroporación (microcentrífuga tapa de llenadoed con 120 l de solución que contiene el plásmido). </li><li> Usando pinzas Dumont curvas transferir una taza de la retina a la cámara de electroporación (por sacar con pala, no presionar), y lo colocan en el interior del electrodo en forma de &#39;U&#39;, con la parte posterior de la retina hacia la parte inferior del electrodo y la lente hacia arriba ( Figura 2D). </li><li> Coloque el ánodo de modo que toque la parte anterior del ojo, al lado de la lente, y el uso de pedal de pie del electroporador entregar los pulsos eléctricos. </li><li> Transferencia de la taza de la retina a electroporación a una nueva placa de Petri con CMF-HBSS y repita el procedimiento de electroporación con cada uno de los vasos de la retina restantes. NOTA: Hasta 6 tazas de la retina puede ser a electroporación con la misma solución plásmido sin una disminución significativa en la eficiencia de la transfección. </li><li> Retire el objetivo y el cuerpo vítreo de cada vaso de la retina a electroporación sujetando con pinzas dentadas de Bonn y tirando suavemente a través de tque por alumno apertura mientras mantiene el ojo en su lugar con el dorso de las pinzas Dumont curvas. </li><li> Proceder a la disociación y la cultura de las células de la retina electroporación siguientes procedimientos estándar. NOTA: Para un protocolo detallado en la disociación y la cultura de células de la retina de pollo se refieren a Vergara y Canto-Soler, 2012, la disposición 4 21 En este protocolo, las células se sembraron a baja densidad en placas de 6 pocillos polyornithine recubiertos o 35. platos -mm (6 x 10 5 células / 35 mm de diámetro, así o plato). El medio utilizado es medio 199 con penicilina / glutamina, 10% de suero de ternera fetal (FCS), y ácido linoleico-BSA a 100 mg / ml. Los cultivos se mantienen en un humidificado 37 ° C incubadora con 5% de CO 2. Usando este procedimiento, 5 - 6 x 10 6 células típicamente se pueden obtener por cada etapa 27 del ojo a electroporación. NOTA: Idealmente, no más de 3 horas debe pasar entre recogida de embriones y de las planchas de células, para garantizar una buena supervivencia celulary minimizar el estrés. Un tiempo típico para la observación y el análisis adicional es después de 4 días de cultivo, ya que en este punto que las células han logrado una buena diferenciación morfológica y molecular y la supervivencia sigue siendo alta. </li></ol></li></ol>

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<li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li>
<li>You, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells%5BTitle%5D)+AND+%22Mol+Biol+Rep%22%5BJournal%5D)">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li>
<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li>
</ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

El paso más crítico para el éxito de este protocolo es la selección de la etapa apropiada de los embriones. En publicaciones anteriores, se da una serie de etapas embrionarias de estas culturas, por lo general definido por los días del día de incubación o embrionarias (ED); por lo que se suele suponer que el uso de ED 5 de ED 6 embriones producirá resultados equivalentes. Sin embargo, hemos encontrado que en el escenario 27 (ED 5), la eficiencia de transfección será de alrededor de 22% de la población total d...

Cultivos de células disociadas de retinas de embriones de pollo se han utilizado ampliamente para estudiar diversos aspectos de la biología de las células fotorreceptoras, incluyendo su supervivencia 2-9 diferenciación 10-12, el crecimiento de neuritas 13, y más. Las ventajas de este sistema, desarrollado en la década de 1980 por Rubén Adler y colaboradores y perfeccionadas por él y para otros grupos de 14-20, residen en las características intrínsecas de la chica como un modelo animal 21. El gran tamaño del ojo polluelo, incluso en las etapas embrionarias, proporciona grandes cantidades de material para cultivos. Por otra parte, cuando los cultivos se realizan con el día embrionario (ED) 5 - 6 retinas, el 55 - 80% de sus células progenitoras diferenciar como fotorreceptores 14,15,18,22,23, y desde aproximadamente el 86% de los fotorreceptores en este animal son conos 24, estas culturas son especialmente adecuados para estudios centrados en este tipo de células. Recientemente hemos desaped y caracterizado una técnica sencilla que permite una alta eficiencia plásmido transfección de las células en estos cultivos, ampliando así la utilidad de este sistema, facilitando estudios missexpression genética 1. El desarrollo de esta técnica se deriva de un vacío en la literatura científica de métodos que proporcionen un nivel aceptable de la transfección para permitir el estudio de la función génica en una célula de manera autónoma. Esto es en parte debido a cultivos neuronales primarios son notoriamente difíciles de transfectar 25,26. Algunas de las técnicas más comúnmente utilizadas previamente disponibles para este propósito incluyen métodos de transfección químicos tales como lipofección o calcio transfección mediada por fosfato, que se traducen en una mayor eficacia en el orden de 3-5% y puede ejercer una toxicidad considerable, 27-32. A pesar de que el uso de plásmidos con un sistema reportero enzimática puede eludir el problema de la pobre eficiencia de la transfección mediante la amplificación de la señal, no lo hacendiscriminar los efectos específicos de la célula, y sus resultados se basan en una población de células pequeñas que pueden no ser representativos del conjunto. Otro método ampliamente utilizado en el pollo, infección por el virus RCAS, sólo es aplicable a las células en proliferación y por lo tanto no es adecuado para este sistema de cultivo primario de la retina 33. En el protocolo actual ojos de embriones de pollo se enucleados en la etapa 27 (ED 5), se retira el epitelio pigmentado de la retina (RPE), y la copa de la retina se coloca en una cámara de electroporación llena con una solución que contiene el plásmido y electroporación utilizando a medida electrodos, seguido por la disociación de la retina y de cultivo utilizando técnicas estándar 21. Después de la optimización de este procedimiento hemos sido capaces de alcanzar consistentemente las eficacias de transfección en el orden del 22% del número total de células en cultivo y 25% dentro de la población de los fotorreceptores solo, sin comprometer las características de supervivencia y diferenciación de lasculturas 1. Aquí proporcionamos un protocolo detallado que describa todos los pasos importantes de este procedimiento a fin de garantizar el éxito y la reproducibilidad de esta técnica.

<table><tbody><tr><td>ECM 830 Electro Square Porator</td><td>BTX/ Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>Genetrode, L-Shaped, 5 mm Gold Tip</td><td>BTX/ Harvard Apparatus</td><td>45-0115 model 512</td><td>Gold tipped electrode used as anode</td></tr><tr><td>Polyimide Tubing</td><td>Vention Medical</td><td>custom made</td><td>Internal Diameter: 0.5 mm/wall thickness: 0.15 - 0.2 mm. Used for insulating gold tiped electrode</td></tr><tr><td>2.5 mm square box filament, 4.5 mm wide</td><td>Sutter Instrument Company</td><td>FB245B</td><td>Used to make cathode electrode</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Gibco - Life Technologies</td><td>14175-095</td><td></td></tr><tr><td>Moloney forceps</td><td>Roboz</td><td>RS-8254</td><td>Serrated; Slight Curve; 4.5&quot; Length</td></tr><tr><td>Dumont tweezers 5/45</td><td>Roboz</td><td>RS-5058</td><td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td></tr><tr><td>Bonn micro forceps, 1x2 teeth</td><td>Roboz</td><td>RS-5172</td><td>Tying Platform; 1x2 Teeth, 0.12 mm Teeth; 3.75&quot; Length; 0.3 mm Tip Width</td></tr></tbody></table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

Los cultivos de fotorreceptores enriquecido cono derivados de las retinas de embriones de pollo se han convertido en una herramienta indispensable para los investigadores de todo el mundo que estudian la biología de las neuronas de la retina, en particular los fotorreceptores. Las aplicaciones de este sistema van más allá de la investigación básica, ya que se pueden adaptar fácilmente a las tecnologías de alto rendimiento para el desarrollo de fármacos. Sin embargo, la manipulación genética de fotorreceptores de la retina en estos cultivos ha demostrado ser muy difícil, que presenta una importante limitación a la utilidad del sistema. Recientemente hemos desarrollado y validado un ex ovo técnica de electroporación plásmido que aumenta la tasa de transfección de células de la retina en estos cultivos por cinco veces en comparación con otros protocolos disponibles en la actualidad 1. En este método los ojos de embriones de pollo se enucleados en la etapa 27, se retira el RPE, y la copa de la retina se coloca en una solución que contiene el plásmido y se electroporó usando Custom- de fácil construcciónelectrodos hechos. Las retinas son entonces disocian y se cultivan utilizando procedimientos estándar. Esta técnica se puede aplicar a los estudios de sobreexpresión, así como a la regulación a la baja de la expresión génica, por ejemplo, mediante el uso de la tecnología del RNAi plásmido impulsado, comúnmente la consecución de la expresión del transgén en 25% de la población de los fotorreceptores. El formato de vídeo de la presente publicación hará que esta tecnología de fácil acceso para los investigadores en el campo, lo que permite el estudio de la función de genes en cultivos de retina primarios. También hemos incluido una explicación detallada de los pasos críticos de este procedimiento para un resultado exitoso y reproducibilidad.

Aquí presentamos un protocolo sencillo para el plásmido transfección en vasos de la retina de pollo enucleados para el cultivo celular disociada subsiguiente. La transfección se consigue mediante electroporación utilizando fácil de hacer electrodos personalizados (Figuras 1 - 2). Los parámetros descritos en este protocolo se han optimizado para obtener eficiencias de transfección que oscilan entre el 20 y el 27% (con un promedio de 22%) (Figura 3D). Tenga en cue...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

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

Cultivos de células de la retina de embriones de pollo constituyen herramientas valiosas para el estudio de la biología de los fotorreceptores. Hemos desarrollado una técnica de transferencia de genes eficiente, basado en ex ovo plásmido electroporación de las retinas antes del cultivo. Esta técnica aumenta considerablemente la eficiencia de transfección a través de protocolos disponibles en la actualidad, lo que hace posible la manipulación genética para este sistema.

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 ...

efficient-gene-transfer-chick-retinas-for-primary-cell-culture

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

8.2K vistas.  The Johns Hopkins University School of Medicine.  Cultivos de células de la retina de embriones de pollo constituyen herramientas valiosas para el estudio de la biología de los fotorreceptores. Hemos desarrollado una técnica de transferencia de genes eficiente, basado en ex ovo plásmido electroporación de las retinas antes del cultivo. Esta técnica aumenta considerablemente la eficiencia de transfección a través de protocolos disponibles en la actualidad, lo que hace posible la manipulación genética p...

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

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 ...

Investigación

Biología del Desarrollo

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurociencias

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 ...

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 ...

Biología

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 ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Transfección de células individuales en embriones de pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...

Transferencia génica eficaz en polluelo retinas para estudios de cultivo celular primario: Un

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