Los autores desean agradecer a Jane Johnson para el Math1, Ngn1, y los anticuerpos Ptf1a y Connie Cepko para el pCAG :: GFP plásmido. Este trabajo fue financiado por el NIH R15 1R15HD059922-01.

1. Los preparativos antes de la electroporación <ol><li> Amplificar el ADN para la electroporación por un máximo de preparación (Prime-It o Qiagen). La concentración del ADN debe ser un mínimo de 1 mg / ml para la absorción eficiente. </li><li> Eliminar 495 l de ADN y se mezcla con 5 l de 0,01% Fast Green en 1 x PBS (tampón fosfato salino) en un tubo de microcentrífuga. </li></ol> 2. Cosecha embrionarias <ol><li> Establecer apareamientos programados de ratones CD-1 (Harlan). Verificar la presencia de tapones vaginales y consideran que la fecha se observa un tapón vaginal como el día embrionario (E) 0,5. Los embriones serán cosechados 11 días después de la visualización de enchufe (E11.5). </li><li> Coloque las herramientas esterilizadas en el 70% de etanol. Tratar una campana de flujo laminar con luz ultravioleta durante al menos una hora antes de su uso. Campana Spray, bandeja de disección, y la disección de alcance con el 70% de etanol. Pre-caliente 1 x PBS a 37 ° C. </li><li> En E11.5 la eutanasia de la hembra de acuerdo a las condiciones aprobadas por el Institutoional Comité para el Cuidado y Uso de Animales (ICCUA). Coloque una bandeja de disección en la campana de flujo laminar. Rociar por el abdomen de la hembra con 70% de etanol. </li><li> Abra la cavidad peritoneal y el pin de las paredes de la bandeja de disección. Sacar los cuernos uterinos de forma que descanse dentro de la cavidad peritoneal. </li><li> Corte cuidadosamente abrir la pared de la trompa uterina. Use una cuchara de 20 mm (Bellas Herramientas Científicas) para eliminar suavemente el embrión en el saco vitelino fuera de la placenta. Embriones Colocar en un plato de cultivo de 100 mm de tejido llena previamente con 10 ml de PBS estéril 1 X que se precalienta en 2,2. </li><li> Repita el procedimiento para todos los embriones presentes. </li></ol> 3. La electroporación de embriones E11.5 (figura 1) <ol><li> Con una cuchara de 20 mm, transferir el embrión primero en un plato nuevo 100 mm precargado con 10 ml de 1XPBS estériles que fue precalentado a 37 ° C. </li><li> Use unas pinzas 11 cm con una punta de 0,05 x 0,02 mm (Bellas Herramientas Científicas) para volver a cuidadose mueven y desechar el saco vitelino. </li><li> Embrión de posición de modo que su cara dorsal quede hacia arriba de manera que se asemeja a los dibujos animados en la Figura 1A. El uso de electrodos de palas 7 mm (Harvard Apparatus) para sostener suavemente el embrión. Los electrodos deben colocarse en cualquier lado del tubo neural en el nivel del ventrículo rombencéfalo cuarto. El ventrículo es visible a simple vista, pero utilizando un microscopio de disección puede facilitar la colocación exacta de pared. Posicionamiento de las paletas es crítico para la determinación de la región que recibe el ADN electroporado. Si el labio inferior rómbica (LIF) del cerebro posterior dorsal embrionaria se desea, las paletas debe ser el lugar de tal manera que están directamente que flanquean la parte más ancha de la apertura del cuarto ventrículo. </li><li> Use una jeringa de 1 cc para elaborar el ADN del plásmido se mezcla con 0,01% Fast Green. 500 l de la mezcla debe ser suficiente para la electroporación de al menos 8-10 embriones (véase 3,5). </li><li> Suavemente la punción de la OV roofplateerlying el cuarto ventrículo con una aguja 25G tuberculina 5/8 unido a la jeringa de 1 cc y se inyecta la mezcla de ADN-colorante en el ventrículo. Inyecciones exitosos se caracterizan por la mezcla de ADN-colorante de llenado del sistema ventricular completo (Figura 1C). La cantidad de mezcla de ADN-colorante inyectado típicamente es menor que 50 l. Las cantidades exactas son variables entre los embriones como una porción de la mezcla tiende a salirse de ventrículo en el PBS que rodea el embrión. Un medio alternativo de la entrega de la mezcla de ADN-colorante sería a través de acceder al sistema ventricular mediante punción del velo que cubre el cerebro medio. De nuevo, las inyecciones exitosas se caracterizan por la mezcla de ADN-colorante de llenado del sistema ventricular completo. </li><li> Entregar cinco pulsos cuadrados con un generador de impulsos eléctricos (BTX) y 7 palas de electrodos mm. Cada impulso es 50 V duradera 5 ms por impulsos con 500 ms entre cada impulso. El tejido cercano al electrodo cargado positivamente entonces Take hasta el plásmido. </li></ol> 4. Cultivo de embriones <ol><li> En una campana de flujo laminar, llenar los pocillos exteriores de una placa de cultivo de 12 pocillos con 2 ml de DMEM/F12 medio suplementado con suero fetal bovino al 10%, 5% de suero equino, 1% de glutamina, 1% de penicilina / estreptomicina que se precalentó a 37 ° C. Las condiciones de cultivo fueron adaptados de los de Diego y sus colegas. 15 </li><li> En embriones de campana de flujo laminar pellizco en abdomen (por debajo del corazón) con unas pinzas y quitar la parte posterior del embrión. Colocar la porción anterior en uno de los pozos llenos de la placa de cultivo de 12 pocillos. </li><li> Repita el procedimiento para todos los embriones. Llenar sólo los pozos exteriores de la placa 12-así para evitar la contaminación de los cultivos. </li><li> Embriones de Cultura en una incubadora a 37 ° C con 5% de CO 2. Expresión de la electroporated plásmido debe ser observable dentro de las 24 h. </li><li> Si ya veces de cultivo se desea, llenar los pozos fuera de una nueva placa de 12 bien con 2 ml delos medios utilizados en 4,1. Con una cuchara esterilizada transferir los embriones a un pocillo de la placa nueva. Coloque de nuevo en la incubadora a 37 ° C. Cultura para un máximo de 48 horas es posible. </li><li> Cuando el tiempo de cultivo deseada, fijar embriones para el análisis (véase más adelante). </li></ol> 5. Preparación de embriones para el Análisis <ol><li> Enjuagar embriones en 1 x PBS a 4 ° C durante cinco minutos. Repita. </li><li> Fijar para el análisis de los embriones en el 2% de paraformaldehído (PFA) en 1XPBS durante 2 horas a 4 ° C. </li><li> Enjuagar embriones en 1 x PBS a 4 ° C durante cinco minutos. Repita. </li><li> Equilibrar los embriones en el 30% de sacarosa en PBS 1 X la noche a 4 ° C. </li><li> Insertar los embriones en la temperatura óptima de corte (octubre) con un compuesto de hielo seco / etanol baño. Los embriones se pueden almacenar a -20 ° C. </li><li> Sección embriones en un criostato (Leica) en secciones de 30 micras y montar en las diapositivas (VWR, Superfrost Plus). Almacenar a -20 ° C. </li></ol> 6. Immunohistochemistry Análisis <ol><li> La inmunohistoquímica se realizó como se describe en 16. Diluciones de anticuerpos primarios utilizados para este estudio incluyen conejo α-GFP (Invitrogen), 1:2500; ratón α-Mash1 (BD Biosciences) 1:100; de conejo α Ngn1-(Johnson Jane) 1:5000; de conejo α-Ptf1a (Jane Johnson ) 1:2500; conejo Math1 (Jane Johnson) 1:100. Incubar corredera plana lado, muestra arriba en una bandeja de tinción a 4 ° C durante la noche. </li><li> Las láminas fueron analizadas en un microscopio compuesto (Olympus BX51). </li></ol> 7. Los resultados representativos Un esquema de la Figura 1A muestra el experimento de electroporación. Figura 1B muestra una vista sagital de un embrión E11.5 antes de la manipulación. El embrión mismo después de la inyección del CAG plásmido que contiene :: GFP en 0,01% Fast Green se muestra en la Figura 1C y un representante embrión no fijada exhibiendo GFP unilateralexpresión en el cerebro posterior dorsal de la cultura 24 horas siguientes se muestra en la Figura 1D. La extensión de la zona de la LIF que se electroporó éxito es variable y parece ser altamente dependiente de posicionamiento de los electrodos. En nuestros estudios se encontró que 52 de 65 (80%) de los embriones electroporated expresado con éxito GFP. El tejido se considerará que se electroporated éxito si era positivo para las buenas prácticas agrarias en las regiones localizadas en varias secciones posteriores a la fijación y el análisis inmunohistoquímico (ver más abajo). Los embriones que no cumplan estos criterios se calificaron como intentos fallidos de electroporación. Una evaluación adicional de la eficiencia de la electroporación puede determinarse mediante la realización de inmunohistoquímica contra GFP en secciones transversales de embriones electroporated a nivel del cuarto ventrículo. Figura 2 muestra representativas de serie de secciones de un embrión mostrando unilateralLa expresión de GFP. Figura 2A muestra un esquema idealizado que ilustra que el lado izquierdo del embrión fue hacia el electrodo positivo. Las secciones transversales (a niveles representados por la historieta medio de la Figura 2A) revelan la expresión de GFP localizada exclusivamente en el lado izquierdo del tejido rombencéfalo (Figuras 2C y 2D). El área electroporado del LIF no se extienden todo el eje antero-posterior del tubo neural como examen de las secciones 300 micras rostral o mayor a la mostrada en la Figura 2C no expresa GFP (Figura 2B). La utilidad de este ensayo para la manipulación de la expresión génica depende de la estabilidad de los dominios de expresión de las proteínas endógenas. El LIF se ha caracterizado como poseedor de dominios únicos progenitoras que se caracterizan por la expresión diferencial de los factores de transcripción proneurales (revisado en 1).Un subconjunto de estos factores (Mash1, Math1, Ngn1 y Ptf1a) fueron elegidos para el análisis debido a su propuesta y / o funciones que se caracterizan en la especificación de precerebellar subtipos neuronales en el LIF, un tema de futuros estudios 16-18. Los cuatro proteínas tienen dominios de expresión muy característicos en el cerebro posterior caudal en E11.5 16-18. Hemos observado que los embriones que fueron colocadas las culturas no aumentan de tamaño y tampoco para iniciar la producción del epitelio del plexo coroideo y la invaginación de la LRL y roofplate, eventos morfológicos que se producen entre E11.5 E12.5 y 5. Basándose en estas observaciones, se determinó que el desarrollo normal en estos embriones se detuvo o se retrasa groseramente y el control comparable para los embriones cultivados debería ser embriones uncultured en E11.5. Para asegurarse de que la cultura y la electroporación no molestar a los niveles de proteínas endógenas, se analizaron cuatro proteínas diferentes immunohistochemistry (IHC) en 34 embriones diferentes que se electroporated y luego se cultivaron durante al menos 24 horas. La Tabla I muestra el número de embriones analizados para cada marcador y el porcentaje de los embriones analizados que mantuvieron niveles normales de la proteína. La Figura 3 muestra representativa de datos IHC a partir de dos de las proteínas analizadas, Mash1 (Figuras 3A y 3B) y Math1 (Figuras 3C y 3D). Hemos observado que la mayoría de los embriones retenidos los niveles normales de expresión después de la electroporación y la cultura (Figuras 3B y 3D) en comparación con los embriones de control en E11.5 (Figuras 3A y 3C). Es importante destacar que los dominios de expresión característicos de estas proteínas no fueron perturbados. <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="Figura 1"/> Figura 1. La electroporación de embriones en E11.5. (A) Esquema de THe experimento electroporación. Un embrión E11.5 se aísla y el plásmido de expresión en 0,01% Fast Green se inyecta en el cuarto ventrículo. El embrión se flanqueada por palas de electrodos y se sometió a un pulso de 50 V antes de ser colocado en cultivo (B) vista sagital de un embrión E11.5 antes de la inyección. (C) El mismo embrión E11.5 después de la inyección del plásmido en 0,01% Fast Green. (D) la expresión unilateral de las buenas prácticas agrarias rombencéfalo observado en embriones E11.5 después de 24 horas de cultivo. MB-mesencéfalo; 4v cuarto ventrículo. <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="Figura 2"/> Figura 2. La expresión de GFP en el tejido electroporated. (A) La caricatura de la izquierda representa la colocación de electrodos en torno a un embrión E11.5. Historieta Medio representa la absorción y la expresión de GFP plásmido que codifica en el lado izquierdo del embrión. Caricatura de la derecha es esquemática de una sección transversal idealizada tomada a través del embrión en levels indicado por las líneas negras en la caricatura de media. (B-D) Inmunohistoquímica para las buenas prácticas agrarias en las secciones transversales a través de un embrión E11.5 electroporado después de 24 horas de la cultura. Las flechas indican la roofplate (RP), que atrapa el anticuerpo secundario. Las imágenes se toman con un aumento de 10 veces. Los niveles relativos de las secciones mostradas están representadas por las líneas horizontales a través de la historieta medio en (A). MB-mesencéfalo; l 4v cuarto ventrículo; rp-roofplate. <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="Figura 3"/> Figura 3. Expresión de proteínas endógenas en el labio inferior rómbico inmunohistoquímica para Mash1 (A y B) o Math1 (C y D) comparando las secciones transversales de embriones E11.5 que no eran cultivadas (A, C) con embriones que se electroporadas con CAG.: : las buenas prácticas agrarias y se cultivaron durante 24 horas (B y D). Imágenes tomadas con un aumento de 10 veces. <table border="1"><tbody><tr><td> Proneurales Transcriptiel análisis factorial </td><td> Número de embriones analizados </td><td> Porcentaje de retención patrones normales de expresión </td></tr><tr><td> Math1 </td><td> 15 </td><td> 86,7% </td></tr><tr><td> Mash1 </td><td> 12 </td><td> 83,3% </td></tr><tr><td> Ngn1 </td><td> 7 </td><td> 71,4% </td></tr><tr><td> Ptf1a </td><td> 6 </td><td> 100% </td></tr></tbody></table> Tabla I. Porcentaje de embriones electroporación y cultivadas de contención normales Dominios proneurales factor de transcripción en el LIF.

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No hay conflictos de interés declarado.

La técnica de electroporación in vitro presentados en este estudio es una nueva metodología que puede ser utilizada de manera eficiente para manipular la expresión génica en embriones de menos de 12 días de gestación. La colocación de los embriones en cultivo permite la expresión del gen introducido y evita la letalidad observada cuando los embriones electroporadas se les permite permanecer en vivo. Esta técnica permite la manipulación de la expresión génica en células progenitor...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-1850 </td><td></td></tr><tr><td> Biologie punta Dumoxel tratado DUMONT fórceps </td><td> Bellas Herramientas Científicas </td><td> 11252-30 </td><td></td></tr><tr><td> 20 mm MORIA perforada cuchara </td><td> Bellas Herramientas Científicas </td><td> 10370-17 </td><td></td></tr><tr><td> ECM 830 Generador de onda cuadrada electroporación </td><td> BTX (VWR) </td><td> 47745-928 </td><td></td></tr><tr><td> Aparato de Harvard 7 Tweezertrodes mm * Electrodos </td><td> BTX (Fisher) </td><td> BTX450165 </td><td></td></tr><tr><td> Fisher Isotemp incubadora de CO 2 </td><td> Pescador </td><td> 1325525 </td><td></td></tr><tr><td> NAPCO de CO 2 del regulador de gas </td><td> Pescador </td><td> 15497020 </td><td></td></tr><tr><td> 12 placas de cultivo tisular </td><td> BD Falcon (Fisher) </td><td> 877229 </td><td></td></tr><tr><td> HyClone líquido Medios DMEM/F-12 (1:1); con L-glutamina y HEPES, 500 ml </td><td> Thermo Scientific (Fisher) </td><td> SH3002301 </td><td></td></tr><tr><td> HyClone * Donante de suero equino </td><td> Thermo Scientific (Fisher) </td><td> SH3007402 </td><td></td></tr><tr><td> Suero bovino fetal, calificado de calor, inactivado </td><td> Invitrogen </td><td> 16140-063 </td><td></td></tr><tr><td> Cellgro * 10.000 UI Penicilina, 10.000 mg / ml de estreptomicina </td><td> Mediatech (Fisher) </td><td> MT-30-002-CI </td><td></td></tr><tr><td> HyClone * L-Glutamina L-Glutamina, 200mm en 0,85% de NaCl </td><td> Thermo Scientific (Fisher) </td><td> SH3003401 </td><td></td></tr><tr><td> Fast-Green </td><td> Pescador </td><td> AC41053-0250 </td><td> 0,01% </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

El labio rómbico es un neuroepitelio embrionario localizado en el cerebro posterior en la unión entre el tubo neural y la roofplate del cuarto ventrículo (revisado en 1). El labio rómbico puede subdividirse en el labio superior romboidal (URL), que abarca rhombomere 1 (R1) y genera las neuronas del cerebelo y el labio inferior rómbica (LIF), que da lugar a diversos linajes neuronales del tronco cerebral 2-4. LRL incluyen los derivados de las neuronas auditivas de los núcleos cocleares y los de los núcleos precerebellar que están involucrados en la regulación del equilibrio y el control del motor 5.8. La neurogénesis de la LIF se produce a través de una gran ventana temporal que abarca embrionarias día (E) 9,5 a 16,5 5, 9. Diferentes linajes neuronales emergen de la LIF, como las células posmitóticas (o nacen) durante distintos días de desarrollo durante esta ventana neurogénica. La electroporación de construcciones de expresión de genes puede ser usado paramanipular la expresión génica en células progenitoras LRL y, potencialmente, puede cambiar el destino de las neuronas que se producen en esta región 10-12. La alteración de la expresión génica de células progenitoras LRL en el ratón mediante electroporación en el útero ha tenido un gran éxito para la manipulación de linajes nacidos en día embrionario E12.5 o 10 más tarde, 12-14. En electroporaciones útero antes de E12.5 no han tenido éxito debido principalmente a la letalidad asociada con la punción de la roofplate cuarto ventrículo, un paso necesario en la entrega de ADN exógeno que se electroporated en el LIF. Sin embargo, muchos linajes LRL derivados se derivan de la LIF anterior a E12.5 9. Estos linajes nacidos a principios incluyen las neuronas que componen el reticular lateral, cuneadas externa, y los núcleos olivar inferior del sistema precerebellar que funcionan para conectar las entradas de la médula espinal y la corteza del cerebelo, 5. Con el fin de manipular la expresión en el LIFde embriones de menos de E12.5, hemos desarrollado un sistema in vitro en el que los embriones se colocan en la cultura después de la electroporación. Este estudio presenta un método eficiente y eficaz para la manipulación de la expresión de los genes de los progenitores LRL en E11.5. Los embriones electroporadas con la proteína verde fluorescente (GFP), impulsado por el promotor CAG ampliamente activa reproducible expresó GFP después de 24 horas de cultivo. Un aspecto crítico de este ensayo es que la expresión de genes sólo se altera debido a la expresión del gen exógeno y no debido a los efectos secundarios que resultan de la electroporación y técnicas de cultivo. Se determinó que los patrones de expresión de genes endógenos permanecen inalteradas en los embriones electroporated y culta. Este ensayo puede ser utilizado para alterar el destino de las células que surgen de la LIF de embriones menores de E12.5 mediante la introducción de plásmidos para la sobreexpresión o tumbar (a través de ARNi) de diferente pro-factores de transcripción neuronales.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Este estudio describe el desarrollo de un<em&gt; In vitro</emTécnica&gt; electroporación, que permite la manipulación de la expresión génica en el labio inferior rómbica mitad de la gestación de los embriones.

En electroporación in vitro del labio inferior rómbico mitad de la gestación de embriones de ratón

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K vistas.  University of Illinois at Springfield. Este estudio describe el desarrollo de un In vitro electroporación, que permite la manipulación de la expresión génica en el labio inferior rómbica mitad de la gestación de los embriones.

Video: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

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 ...

Investigación

Neurociencias

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

La cuantificación de la actividad de Cis Elementos reguladores en la retina del ratón por electroporación explante

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Del ratón en el útero de electroporación: Transefection controlada Gene espacio-temporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferencia de genes a la oreja del ratón en desarrollo por el interior<em&gt; En Vivo</em&gt; La electroporación

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

La electroporación del Rombencéfalo trazar trayectorias axonal y metas sinápticas en el embrión de pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

La manipulación genética del ratón en desarrollo a través de Hipotálamo<em&gt; En el útero</em&gt; La electroporación

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Un<em&gt; In-vitro</em&gt; Preparación de las neuronas entéricas aisladas y células gliales del plexo mientérico del ratón adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.