Damos las gracias a Humana Press el permiso para publicar la pipeta de microinyección de fabricación de la figura que apareció originalmente en la página 130 de la referencia 11, Larry Dlugas y Wong Steven, OHSU Departamento de Comunicación Educativa, por videografía, Larry Dlugas para el diseño y edición de vídeo, Adam M. S Quinn, el diseñador senior, Trion / Envirco para el diseño de nuestra costumbre campana de flujo laminar horizontal, y el orfebre de Les para proporcionar el esquema técnico, Víctor Monterroso, MV, MS, PhD y Chatkupt Tom, DVM, OHSU Departamento de Medicina Comparada, de orientación con nuestro animales protocolo de atención, las técnicas quirúrgicas, y profilácticos régimen de analgesia; Marcel Perret-Gentil, DVM, MS, por compartir su folleto sobre técnicas de sutura veterinarios; Edward Porsov, MS, para el diseño de nuestra Adobe Premiere Pro microscopio de vídeo monitor y la computadora, y Blanca Lía y Jonas Hinckley de la LNS subtítulos (Portland, Oregón). Este trabajo fue apoyado por subvenciones del Instituto Nacional de la Sordera y other Trastornos de la Comunicación: 008595 DC R01 y R01 DC 008595-04S2 (JB) y P30 DC005983 (Oregon Audiencia Centro de Investigación Core Grant, Peter Gillespie, investigador principal).

1. La laparotomía ventral <ol><li> Anestesie una presa cuyos embriones se encuentran en el día embrionario 11.5 (E11.5; mediodía del día se detecta un tapón vaginal es el día del desarrollo embrionario 0,5) por inyección intraperitoneal de una solución de pentobarbital sódico anestesia (7,5 l por cada gramo de peso corporal). Solución de trabajo anestésico: 180 l de 50 mg / ml de solución de pentobarbital sódico; 100 l de etanol absoluto; 320 l de 65 mg / ml de sulfato de magnesio acuoso (modula el tono uterino), y 400 l de propilenglicol (miscibles con vehículo acuoso y orgánico componentes). </li><li> Evaluar la integridad de la anestesia mediante la realización de las pruebas de estímulos nocivos: apretar la pata, la respuesta y abrir y cerrar de tocar la mejilla y vibrisas; pizca cola. Aplique una pomada oftálmica estéril de las córneas. </li><li> Afeitarse la piel abdominal de la zona suprapúbica de la caja torácica con tijeras y una hoja fina (Oster # 40 hojas). Desinfecte el abdomen con etanol al 70%, 10% de yodo povidona (Betadine), unnd 70% de etanol, de forma secuencial. Coloque el abdomen del ratón hacia abajo en un campo estéril, a continuación, establecer una almohadilla térmica o una placa caliente (37 ° C) durante 2-5 minutos. </li><li> Incisión en la piel del abdomen en la línea media ventral, con la pelota de punta de las tijeras. Extienda la incisión de 10-14 mm. Identificar la línea alba, una banda de avascular, blanco tejido conectivo a lo largo de la línea media ventral de la pared abdominal. Incisión en la línea alba con la pelota con punta de tijera y extender la incisión 10-14 mm. Inmediatamente irrigar el abdomen con precalentado (37 ° C) solución de Ringer lactato. </li><li> Externalizar los dos cuernos del útero con una pinza de anillo, sin ejercer demasiada presión sobre los lugares de implantación. Lavar los cuernos uterinos con externalizados precalentado, solución de Ringer lactato. </li></ol> 2. Microinyección Transuterine <ol><li> Se fabrica una pared gruesa, pipeta de vidrio de borosilicato capilar de microinyección con las siguientes características: 12-16 μm diámetro exterior y un bisel de 20 grados. En un extractor de Sutter P-97 pipeta con filamento de caja pequeña, utilice el siguiente programa: el calor = prueba de rampa más 3 unidades, la presión = 200; tire = 0; velocidad = 46; tiempo = 100). Con el Sutter BV-10 biselador, utilice el 104C (oro) disco abrasivo para pipetas de gran diámetro. Volver a suspender el plásmido en la expresión g 3-4 / l de fosfato de calcio libre amortiguada (pH 7.2 a 7.4). Añadir verde rápido cristalina del ADN concentrada vórtice, suavemente durante 30 segundos, y girar a 10.000 g durante 10 segundos. La cantidad mínima de verde rápido requerido para rastrear visualmente la eficacia de la inyección se determina empíricamente. Rellene la pipeta biselado con el ADN / solución verde rápido. Conecte la pipeta de microinyección cargado al titular de la pipeta de la presión de inyección (Picospritzer con&gt; 99% de nitrógeno puro como gas de la fuente). </li><li> Transiluminar el útero con baja intensidad, luz halógena para visualizar el embrión dentro de su lugar de implantación. Identificar la beating corazón, vesículas cerebrales, yemas de las extremidades, la naciente 4 º ventrículo del cerebro posterior, y los ojos. Riegue el útero cada 2 minutos con precalentado, la solución de Ringer lactato para mantener la hidratación. Aplique una leve presión sobre el útero para reorientar el embrión e identificar los puntos de referencia anatómicos se ha indicado anteriormente. </li><li> Orientar el embrión para identificar la vena de la cabeza primaria cuya anterior y posterior del flanco sucursales en el territorio mesenquimales en la que el otocyst reside. El otocyst apropiada no puede ser vista por transiluminación del útero. El otocyst se encuentra a medio camino entre las ramas anterior y posterior de la cabeza de la vena principal que, junto con el tronco principal de la vena, constituyen la forma de los montantes o los postes, en un campo de fútbol americano. El otocyst está a medio camino entre los montantes. </li><li> Inserte la pipeta de inyección a través del útero de una trayectoria en línea con la ubicación de la presunta otocyst. Pulso del microinyector una vez después de pasar por el utera visualizar el colorante trazador y la aproximación de la localización de la punta de la pipeta. Avanzar en la pipeta bajo control micrométrico y el pulso de nuevo para evaluar la profundidad. Además avanzar en la pipeta en el mesénquima de la cabeza lateral y el pulso en varias ocasiones. El éxito de la focalización otocyst revelará la forma cónica del conducto endolinfático dorsal y la concha de abanico en forma de concha del vestíbulo. Libere la presión sobre el útero, retire la pipeta del embrión / útero en un solo movimiento, e inmediatamente regar el útero con precalentado, solución de Ringer lactato. </li></ol> 3. Electroporación in vivo <ol><li> Riegue el útero con una solución de lactato de Ringer. Solución de Ringer lactato recién aplicado es necesario par eléctricamente los electrodos de estilo de paletas en el útero. Humedecer las superficies de los electrodos de tungsteno con el Ringer lactato. Centro de la otocyst inyecta en el camino de los electrodos. Suavemente comprimir el útero con la Autoridad Palestina electroporaciónddles. El cátodo está en contacto con la pared uterina lateral a la otocyst inyectado y el ánodo está en contacto con la pared uterina adyacente a la otocyst no inyectado. Dispare un tren de ondas de pulso cuadrado con el interruptor de pedal en el electroporador. Electroporación parámetros son los siguientes: 5, 50 ms pulsos a 43 voltios por impulso y una demora 950 milisegundos entre impulsos. Lavar inmediatamente el útero después del tren de pulsos se entrega. Anotar la corriente suministrada al tejido: 60-100 mAmps es suficiente para transfectar óticas progenitores epiteliales. Inyectar y electroporar 4-6, embriones E11.5 por la presa. </li><li> Realizar una microinyección segundo, independiente de la transuterine acuosa de dextrano fluorescente (Alexa Fluor 488 si el plásmido de expresión codifica una proteína fluorescente de color rojo o Alex Fluor 594 si el plásmido de expresión codifica una proteína fluorescente verde) en el ventrículo del 4 de esos embriones cuyos oídos interno son precisión inyectado y por lo menos 60 mAmps de corriente por pulso era delivered durante electroporación. El dextrano fluorescentes serán detectables en el nacimiento en el cerebro posterior y permitir la selección de las crías cuyos oídos internos fueron manipulados durante la embriogénesis (ver 3.5). </li><li> Lavar los cuernos uterinos con solución de Ringer lactato. Vuelva a insertar los cuernos uterinos en la cavidad abdominal. Enjuague la cavidad uterina con 2-4 ml de precalentado, solución de Ringer lactato y permitir que el desbordamiento de drenaje de la zona de la incisión en el campo estéril. Vuelva a colocar el drapeado con el material seco y estéril. Suturar la pared abdominal con una aguja no cortante y una sutura reabsorbible 6-0. Nosotros preferimos una puntada corriente, el bloqueo de cada puntada otro, tanto para la pared abdominal y la piel. </li><li> Seque la piel de las presas y administrar un fármaco no esteroide anti-inflamatorio, como meloxicam por vía subcutánea. Regreso de la presa a la jaula de recuperación precalentado en un campo estéril. Controlar y registrar las respiraciones de las presas, la apertura del sitio de la incisión y la vagina de secreción sanguinolenta. El sangrado es ravolver, pero si está presente y sin cesar, la eutanasia a la presa, mientras que ella todavía está bajo anestesia. Tenga en cuenta el tiempo que recupera la conciencia y los intentos de caminar. Vuelva a colocar el dique de la colonia de ratones cuando muestra signos de comer y beber y ha empezado a construir nidos. Típicamente, esto se produce dentro de las 12 horas. </li><li> Al momento de nacer (después del día 0), el flash de la región posterior del cerebro de cada cachorro de la camada para detectar la fluorescencia dextrano usando un microscopio de disección stereofluorescence con las buenas prácticas agrarias o Texas filtro rojo establecer, según corresponda. Volver a la presa de lactancia sólo los cachorros que muestran el etiquetado posterior del cerebro. </li></ol> 4. Los resultados representativos <img src="/files/ftp_upload/3653/3653fig1.jpg" alt="Figura 1" /> Figura 1. La electroporación mediada por la transferencia de genes para el desarrollo de la cóclea. El otocyst E11.5 fue inyectado con un plásmido que codifica la expresión de una mayor proteína verde fluorescente (EGFP) y electroporated (pulso traen los parámetros de: cinco, 43 pulsos voltios a 50 ms / pulso y 950 ms de retardo entre impulsos). A) Un representante del oído interno de un día de vida 6 (P6), cuya cría otocyst se inyectó y electroporated en E11.5 demostrando EGFP expresión de la base a través de la vuelta media de la cóclea. La pared lateral de la cóclea fue retirado de la vuelta media y el ápice solamente. Progenitores E11.5 que dan lugar a la cúspide no fueron transfectadas. B) Todo el montaje inmunoticción de la cóclea (A) con el marcador de células de pelo, 7 bis, la miosina (Myo7a), indica que la expresión de EGFP sigue la trayectoria del órgano de Corti y es grande localizado en la célula de pelo de soporte de epitelio sensorial. C) Un representante del oído interno de un embrión E18.5 cuya otocyst se inyectó y electroporated en E11.5. La proyección de láser confocal demuestra la expresión de EGFP en Myo7a-positivas las células ciliadas sensoriales. D) láser confocal de proyección del epitelio sensorial coclear del órgano de Corti E18.5 indica EGFP expression en las células ciliadas internas (IHC), las células ciliadas externas (OHC), las células del interior de phalangial (IPC), las células de los pilares (pc), y las células de Deiters (DC). La barra de escala en (B) se aplica a (A).

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La transferencia de genes en el oído interno del ratón en desarrollo: La oreja de ratón interno se desarrolla a partir de la placoda ótica durante la primera semana de desarrollo después de la implantación 12,13. Al día embrionario 9.5 (E9.5), la placoda se invagina y se transformó en una vesícula llena de líquido llamado otocyst 2. Otic precursores en la vesícula dar lugar a las células sensoriales y sensorial dentro del oído interno madura, así como las neuronas que...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Micro Case esterilización </td><td> Roboz </td><td> RS-9900a </td><td> 8X8.5X1.25 pulgadas </td></tr><tr><td> Con punta de bola tijera </td><td> Herramientas de Bellas Ciencia </td><td> 14109-09 </td><td></td></tr><tr><td> Pinzas de anillo </td><td> Herramientas de Bellas Ciencia </td><td> 11106-09 </td><td> 4.8mm OD ID/6mm </td></tr><tr><td> Pinza Adson tejido </td><td> Herramientas de Bellas Ciencia </td><td> 11027-12 </td><td></td></tr><tr><td> Aguja del conductor </td><td> Herramientas de Bellas Ciencia </td><td> 12502-12 </td><td></td></tr><tr><td> Alergia bandeja de Jeringa </td><td> Becton Dickison </td><td> 305536 </td><td></td></tr><tr><td> Sutura 6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0,7 sutura gastrointestinal métrica </td></tr><tr><td> Ringer lactato inyectable USP </td><td> Baxter </td><td> 2B2323 </td><td></td></tr><tr><td> Verde rápido </td><td> Sigma Aldrich </td><td> F7258 </td><td></td></tr><tr><td> Borosilicato capilar de vidrio </td><td> Aparato de Harvard </td><td> 30-0053 </td><td></td></tr><tr><td> Solución de sodio Nembutal </td><td> Ovation Pharmaceuticals Inc. </td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific </td><td> M63-500 </td><td></td></tr><tr><td> Propileno glicol </td><td> Fisher Scientific </td><td> P355-1 </td><td></td></tr><tr><td> Etanol </td><td> Sigma Aldrich </td><td> E7023-500 </td><td></td></tr><tr><td> El meloxicam </td><td> Boehringer Ingeheim </td><td> NADA 141-219 </td><td></td></tr><tr><td> Micropipeta Extractor </td><td> Sutter Instruments </td><td> P-97 </td><td> FB255B filamento de caja, consultar libro de cocina con una pipeta de los instrumentos de Sutter </td></tr><tr><td> Microelectrodo Biselador </td><td> Sutter Instruments </td><td> BV-10 </td><td> El disco biselado 104C para pipetas de gran tamaño; consulte el manual del propietario para la teoría de biselado </td></tr><tr><td> Micropipeta titular de la </td><td> Warner Instruments </td><td> MP-S15T </td><td> Para una pipeta de 1,5 mm de diámetro exterior y el puerto de presión hembra para tubería de Picospritzer. </td></tr><tr><td> Pinzas de estilo de electrodos </td><td> Protech International Inc. </td><td> CUY650P5 </td><td> 5 mm de diámetro exterior </td></tr><tr><td> De onda cuadrada Electroporador </td><td> Protech International Inc. </td><td> CUY21EDIT </td><td> Pedal recomendado </td></tr><tr><td> PICOSPRITZER III </td><td> Parker Hannifin </td><td> 051-0500-900 </td><td> Pedal recomendado </td></tr><tr><td> Micromanipulador de control manual </td><td> Aparato de Harvard </td><td> 640056 </td><td></td></tr><tr><td> Laminar horizontal banco de flujo limpio </td><td> Envirco </td><td></td><td> Modificaciones personalizadas en LF 630-10554. Ver información complementaria para el esquema de la campana. </td></tr><tr><td> Leica stereofluorescence disección microcope con Lumencor SOLA motor de la luz </td><td> Bartels y Stout y Lumencor </td><td> MZ10F con Lumencor SOLA motor de la luz </td><td> Pedales para enfocar el MZ10F y para activar el motor de la luz se recomienda SOLA </td></tr><tr><td> Alexa Fluor 594 Dextrano </td><td> Invitrogen </td><td> D22913 </td><td> Acuosa 10mg/ml, </td></tr><tr><td> Alexa Fluor 488 Dextrano </td><td> Invitrogen </td><td> D22910 </td><td> Acuosa 10mg/ml, </td></tr><tr><td> Enviro-dri </td><td> Pastor Especialidad de Papanicolaoures </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

El oído interno tiene 6 epitelios de mamíferos distintos sentidos: 3 crestas de las ampollas de los canales semicirculares, máculas en el utrículo y el sáculo y el órgano de Corti en la cóclea en espiral. El crestas y máculas contienen células ciliadas vestibulares que transduce estímulos mecánicos para servir al especial sentido del equilibrio, mientras que las células auditivas ciliadas en el órgano de Corti son los transductores primarios para escuchar 1. Especificación del destino celular en estos epitelios sensoriales y la morfogénesis de los canales semicirculares y la cóclea llevará a cabo durante la segunda semana de gestación en el ratón y son en gran parte completada antes del nacimiento de 2,3. Estudios sobre el desarrollo del oído interno del ratón se realizan rutinariamente por la recolección de embriones transgénicos en diferentes etapas embrionarias o postnatal para comprender mejor la base molecular de la telefonía celular y / o fenotipos morfológicos 4,5. Se postula que la transferencia de genes a la oreja de ratón en desarrollo interno en el útero &lt;/ Em&gt; en el contexto de la ganancia y la pérdida de la función de los estudios representa un enfoque complementario a la transgénesis de ratón tradicional para el interrogatorio de los mecanismos genéticos que subyacen al desarrollo de mamíferos oído interno 6. El paradigma experimental para llevar a cabo los estudios de genes misexpression en el oído interno del ratón en desarrollo ha demostrado aquí se resuelve en tres pasos generales: 1) laparotomía ventral, 2) la microinyección transuterine, y 3) electroporación in vivo. Laparotomía ventral es una técnica de supervivencia de los ratones quirúrgica que permite la exteriorización del útero para tener acceso experimental a los embriones implantados 7. Microinyección Transuterine es el uso de biselados micropipetas capilares de vidrio, para introducir el plásmido de expresión en el lumen de la vesícula ótica o otocyst. In vivo electroporación es la aplicación de onda cuadrada, impulsos de corriente directa para conducir la expresión del plásmido en las células progenitoras 8-10. 11. Ratón embriológicos técnicas experimentales pueden ser difíciles de aprender de la prosa y las imágenes fijas solamente. En el presente trabajo, demostramos los 3 pasos en el procedimiento de transferencia de genes. La mayoría de la crítica, hacemos uso de la microscopía de vídeo digital para mostrar con precisión cómo: 1) identificar la orientación del embrión en el útero, 2) reorientar los embriones para la orientación inyecciones a la otocyst, 3) microinyectar ADN se mezcla con solución de tinte trazador en el otocyst en los días de embriones 11.5 y 12.5, 4) electroporar la otocyst inyecta, y 5) los embriones de la etiqueta electroporadas para la selección después del parto en el nacimiento. Proporcionamos ejemplos representativos de éxito transfectadas oído interno: una guía ilustrada de las causas más comunes de la mala focalización otocyst, discutir cómo evitar los errores comunes de metodología y las directrices actuales para la redacción de una g en el úteroeno animales transferencia de la atención de protocolo.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

La oreja de ratón interno es un órgano sensorial placoda derivada del desarrollo cuyo programa se elaboró ​​durante la gestación. Se define un<em&gt; En el útero</emGen&gt; transferencia de la técnica que consiste en tres pasos: laparotomía ventral del ratón, la microinyección transuterine, y<em&gt; En vivo</em&gt; Electroporación. Utilizamos microscopía de vídeo digital para demostrar los críticos técnicas embriológicas experimentales.

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

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

13.9K vistas.  Oregon Health & Science University. La oreja de ratón interno es un órgano sensorial placoda derivada del desarrollo cuyo programa se elaboró ​​durante la gestación. Se define un En el útero transferencia de la técnica que consiste en tres pasos: laparotomía ventral del ratón, la microinyección transuterine, y En vivo Electroporación. Utilizamos microscopía de vídeo digital para demostrar los críticos técnicas embriológicas experimentales.

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gen de entrega de cerebro de rata postnatal por la inyección no ventricular plásmido y electroporación

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Investigación

Neurociencias

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 ...

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Aislar las células que expresan LacZ de tejidos de ratón del oído interno mediante citometría de flujo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imágenes de alta resolución en vivo del comportamiento de las células en el desarrollo de neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Grabaciones de patch clamp en Inner células del oído pelo Aislado de pez cebra

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método quirúrgico para la entrega de genes mediada por virus de la oreja del ratón Interior a través de la ventana de membrana Ronda

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...