Este trabajo fue financiado por RIKEN Brain Science Institute (BSI), Programa de Ciencias Humanas Frontera (HFSP) (adjudicado a TS) y RIKEN Programa de Investigación Asociado Juvenil (JRA, adjudicado a MK) RIKEN Brain Science Institute (BSI), Programa de Ciencias Humanas Frontera (HFSP) (adjudicado a TS) y RIKEN de Investigación Programa Junior Associate (JRA, adjudicado a MK) financió este trabajo.

1. Preparación de la solución capilar y de ADN para la inyección <ol><li> Purificar el ADN plásmido con un Maxi-prep o método equivalente, y hacer una concentración final de 2.3 mg / l. Alícuota de 100 ug de ADN, añadir 10μl de tinte verde rápido (1% de acciones) y TE (10 mM Tris base, 1 mM EDTA, pH 8.0) y ajustar un volumen de 100 l para la concentración final de ADN para la mezcla de la inyección de 1 g / l. La concentración de ADN del plásmido se puede cambiar depende del tamaño de un plásmido, y también su eficiencia de transfección, que debe ser evaluado individualmente. </li><li> Haga girar la solución de ADN durante 5 minutos a 14.000 rpm a temperatura ambiente y recoger el líquido sobrenadante para eliminar cualquier residuo. </li><li> Tirar de un capilar de vidrio de tubos de vidrio tubea capilar mediante un extractor de micropipeta. Pellizcar la punta con una pinza para que la punta tiene un diámetro de 20-30 micras. De 800 m-1 mm de la punta y comprobar el diámetro exterior es inferior a 60 micras (Fig. 1A). </li><li> Conecte el capilar para el micromanipulador, y rellenar con aceite mineral. Para llenar el capilar con la solución de ADN, sumergir la punta en la solución y aspirar la solución de ADN. </li></ol> 2. Palo electrodos de platino Aquí usamos electrodos revestidos de platino (Nepe gen, CUY611P2-1) para electroporar en la región superficial del cerebro, tales como la corteza (Fig. 1F). 3. Preparación de micro-electrodos El uso de micro-electrodos en electroporated en partes profundas del cerebro (es decir, el tálamo y el hipotálamo) (Fig. 2B-E). <ol><li> Un extremo del tungsteno (para los electrodos negativos) y platino (para electrodos positivos) se enrolla en alambres dorados alfileres y se fija con parafilm. Inserte el cable a la madre de un hisopo de algodón y envolver los dos extremos con parafina (Fig. 1B). </li><li> Uniformemente afilar la punta del alambre con papel de lija hasta que llega a 20-30 m de diámetro exterior y 60 m de 800 m-1 mm de la punta (Fig. 1C y D). </li><li> Aplicar una fina capa de esmalte de uñas con el cable para el aislamiento. Después de que el esmalte se haya secado, use un hisopo de algodón empapado en acetona para quitar de la punta (unos 200 m de la punta). Nota importante: Con el fin de evitar daños en el útero del ratón, la parte de dentro de 800 m-1 mm de la punta no debe ser mayor de 70 micras de diámetro (Fig. 1E). </li></ol> 4. Preparación de la cirugía <ol><li> Anestesiar a un ratón embarazadas (E9.5-E15.5). Demostramos anestesia con grado farmacéutico pentobarbital sódico (Cuadro II, 50 microgramos por gramo de peso corporal, por vía intraperitoneal se inyectan y esperar 5 min). Alternativas al pentobarbital sódico como anestésico incluyen la inyección de ketamina / xilazina o anestésicos gas (gas es preferible para los procedimientos de corta duración). La inyección se debe hacer con atención para evitar que cualquiera de los órganos, de lo contrario será causa de daños en el útero o matar a los animales. Cuando la propiedad intelectual se lleva a cabo con éxito, la tasa de supervivencia es superior al 95%. Asimismo, la cirugía se puede realizar al aire libre en el medio ambiente. Antes de iniciado el funcionamiento, prueba de la falta de respuesta por parte del animal a pellizcar sus dedos para confirmar la anestesia. </li><li> Afeitar el vello del abdomen con una cuchilla de afeitar y un 50% EtOH. Preparar la piel con la alternancia de matorrales de Betadine y alcohol (70%). </li><li> Hacer una incisión en la línea media abdominal con tijeras finas y sacar todos los cuernos uterinos con cuidado en un 37 ° C precalentado tampón fosfato salino (PBS) una gasa humedecida de algodón, que se coloca alrededor de la herida. Mantener la humedad del útero con PBS todo el tiempo. </li><li> Mantenga un cable flexible de fibra óptica entre los dedos índice y medio y lo coloca en el cuerno uterino. Cable limpio con EtOH al 70% antes de su uso. No microscopio o lupa es necesario para la visualización. Posición del útero entre el cable de luz de fibra óptica óptica y el pulgar, y apretar suavemente para empujar hacia arriba el embrión más cercano a la pared uterina. </li></ol> 5. La inyección de ADN y la electroporación <ol><li> Al embrión se coloca, inserte con cuidado capilar de vidrio en el ventrículo objetivo e inyectar aproximadamente 1 l de solución de ADN (Fig. 2A). </li><li> Para la electroporación a la parte superficial de la brainIf usar con electrodos de platino palo para electoporation, colocar los electrodos fuera del útero y aplicar sugerido de onda cuadrada de impulsos de corriente de acuerdo a las instrucciones de fabricación (Tabla I). Si electroporación usingFor a una mayor parte del cerebro, la electroporación nmicro, electrodos Eedle tipo se utilizan. Iinsert un electrodo de tungsteno bien negativos en el ADN inyectado ventrículo y un electrodo de platino en el útero y la región de destino entre dos electrodos, a continuación, aplicar sugerido de onda cuadrada de impulsos de corriente (Tabla I) (Fig. 2B). </li></ol> 6. Después de la electroporación <ol><li> Vuelva a colocar el cuerno uterino en su origubicación con INAL y añadir 500 l de 37 ° C pre-calentado PBS. </li><li> Suturar la capa interna de sutura quirúrgica y la capa externa cercano con una 9 mm clip de autoclipauto. </li><li> Animales a cabo en un cojín de la calefacción durante 2 horas para permitir la recuperación de la anestesia. Analgésicos como el carprofeno o buprenorfina puede ser administrada para el dolor post-operatorio. </li></ol> 7. Los resultados representativos: Algunos ejemplos de electroporación cortical de pCAG-EYFP construir con electrodos revestidos de platino en diferentes puntos de tiempo se muestra en la figura Figura 32. Los cerebros son electroporated en E13.5, E14.5 y E15.5, y se cosecha en el día postnatal (P) 6. AEl tinción ntibody de RORC muestra la posición de la capa 4 y la posición de las células transfectadas EYFP son reveladas por tinción de anticuerpos GFP, y ambas imágenes son superpuestas (Fig. 3 A y B). Etiquetados células corticales son capa de células corticales electroporación es claramente diferente en cada experimento (Fig. 32), lo que sugiere que dependen del tiempo de electroporación hace cortical capa específica de GOF / LOF posible. La viabilidad de los embriones y la eficiencia de la transfección de células varía en función de la edad del embrión y la ubicación de la electroporación. Las muestras se muestran en la Tabla 1, sin embargo, las condiciones deben ser optimizados para cada electroporación dependiendo de la etapa y la parte del cerebro. Electroporación en un tejido profundo, como el tálamo y el hipotálamo con el uso de electrodos revestidos de platino a menudo dan de baja eficiencia (Tabla 1). Este problema se puede resolver mediante el uso de micro-electrodos whichelectrodes, que llegará a las partes profundas del cerebro. Figura 3 4 ejemplo muestra de pCAG-EYFP electroporación en el diencéfalo en desarrollo. Solución de ADN plásmido se inyecta en el ventrículo 3 º en E11.5 y seguido por micro electroporación (Fig. 3C4C). El área está limitada por electroporación en el tálamo, donde la mayoría de los proyectos axones a la corteza (Fig. 3B4B). Proyección axonal al nivel 4 de la corteza también puede ser visualizado por EYFP, que fillesfills a toda la neurona. (Fig. 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Figura 1"/> Figura 1. Esquema de dibujos animados que demuestran la forma correcta de los capilares. (A) Un tubo capilar de vidrio se tira con un extractor micropipeta para hacer cierta forma. Un consejo es fuera aplastado por unas pinzas para que sea 20-30 micras. De 800 m-1 mm de la punta (el soporte de color rojo) y asegúrese de que el diámetro exterior es inferior a 60 micras (soporte de color verde). (B) Un extremo del tungsteno o alambres de platino se enrolla sobre chapado en oro alfileres y se fija con parafilm. A continuación, utilice papel de lija para que su punta para convertirse en diámetros de 20 a 30 micras externa y 60 micras de 1 mm de la punta. (C) Imagen del alambre de tungsteno antes de darles forma. (D) de la imagen del alambre de tungsteno después de darles forma. (E) de la imagen del alambre de tungsteno después de aplicar la capa delgada de esmalte de uñas. Barra de escala en E es de 10 micras (más pequeño) de la CE. (F) Electrodos de platino (gen Nepa. CUY611P2-1). Barra de escala en la F es de 2 mm. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Figura 2"/> Figura 2. Caricatura esquema de procedimiento quirúrgico. (A) Diagrama de la inyección en el ventrículo lateral o embriones de gran tamaño (lado izquierdo) y en 3 º ventrículo o embriones pequeños (lado derecho). (B) Diagrama de electrporation por electrodo de platino palo (lado izquierdo) y por la aguja del electrodo tipo (lado derecho). <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Figura 3"/> Figura 3. Desarrollo de ratón telencéfalo se electroporated con pCAG-EYFP en E13.5 (A), E14.5 (B) y E15.5 (C) y se cosecha a P6. Los cerebros fueron seccionados coronal y se tiñeron con anticuerpos GFP o un anticuerpo RORC por separado y las imágenes se funden. (A) Electroporación de pCAG-EYFP plásmido en E13.5 transfectar muchos de 4 capas de neuronas. que se superponen con una capa de 4 marcadores RORC. (B) Electroporación de pCAG-EYFP plásmido en las células E14.5 transfectar, que son más superficiales que la capa 4 neuronas. (C) E15.5 electroporación etiqueta de muchas neuronas superficial como la capa 2 / 3. Barra de escala 200 micras. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Figura 4"/> Figura 4. Transfección de EYFP en los axones tálamo-corticales (TCA). (A) terminal de TCA en la capa cortical 4 se pone de manifiesto mediante la tinción de anticuerpos GFP. Tinción RORC anticuerpos se utiliza para mostrar una posición de la capa 4. Ambas imágenes están tomadas de la misma sección y se fusionaron. (B) Posición de la electroporación en el tálamo también se muestra en la tinción de anticuerpos GFP y la tinción de RORC, cuya expresión está restringida en el tálamo 11. (C) Diagrama de dibujos animados para la electroporación y la proyección del tálamo TCA se muestran. Barra de escala 200 micras.

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	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

No hay conflictos de interés declarado.

En este protocolo, sólo se describen los beneficios de la técnica de la transfección pCAG-EYFP en la zona restringida de un pequeño cerebro de embriones utilizando diferentes electrodos en forma. Para la comparación de la eficiencia de transfección por el promotor diferente se ha descrito antes, lo que demuestra la especificidad de células tipo 1. Sin embargo, existen limitaciones para la técnica, tales como la transfección es transitorio y varía depende del plásmido, que no tiene ningún tipo de especificidad celular y es difícil controlar el número de células transfectadas. ThereforeHowever, la combinación de este protocolo con otras técnicas aún más las posibilidades de ofrecer diferentes métodos de manipulación. En primer lugar, la incorporación de promotor de células específicas en el ADN de plásmido permite la transfección de células de tipo específico, como neuronas, células gliales y astrocitos. En segundo lugar, ya que la transfección es transitorio, no es adecuado para los científicos que quieren analizar la función del gen en la vida posterior. Por lo tanto, la combinación con una transposasa para hacer el ADN plásmido integrarse en el genoma va a convertir en una transfección estable 8. Adopción que viene, de la tet-on o sistema tet-off será posible manipular el timing o tipo de células específicas de expresión génica en las células neuronales 8. Alternativamente, se puede combinar este protocolo con el tamoxifeno-inducible Cre-ER (T) recombinasas 9 y ratones reportero de 10. Esta amplia accesibilidad de los tejidos drásticamente cambian los diseños experimentales utilizados en la neurociencia.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> tungsteno diámetro del alambre desnudo 125 m </td><td> Sistemas AM </td><td> 797600 </td></tr><tr><td> platino desnuda diámetro del alambre de 125 micras </td><td> Sistemas AM </td><td> 767000 </td></tr><tr><td> Electrodos revestidos </td><td> Nepa gen </td><td> CUY610P4-1 </td></tr><tr><td> capilar de vidrio del tubo </td><td> Stoelting </td><td> 50611 </td></tr><tr><td> micromanipulador </td><td> KD Científico </td><td> KDS310 </td></tr><tr><td> tijeras </td><td> Roboz </td><td> RS-5865 </td></tr><tr><td> generador de impulsos </td><td> AM Sistemas </td><td> Modelo 2100 </td></tr><tr><td> 9 mm autoclip </td><td> Roboz </td><td> RS-9260 </td></tr><tr><td> chapado en oro alfileres </td><td> WPI </td><td> 5482 </td></tr><tr><td> RORC anticuerpos </td><td> Perseo Proteómica </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

A fin de comprender la función de los genes expresados ​​en la región específica del cerebro en desarrollo, incluyendo las moléculas de señalización y las moléculas axón orientación, la transferencia de genes locales o knock-out es obligatorio. La orientación de genes knock-in o knock-out en las regiones locales es posible llevar a cabo con la combinación con una línea específica CRE, que es laborioso, costoso y consume mucho tiempo. Por lo tanto, un método de transfección simple, una técnica en el útero de la electroporación, que se puede realizar con poco tiempo, será muy útil para probar la posible función de los genes candidatos antes de la generación de 1,2 animales transgénicos. Además de esto, en el útero de la electroporación se enfoca en áreas del cerebro donde no existe línea específica CRE, y limitará la letalidad embrionaria 3,4. A continuación, presentamos un método de electroporación en el útero la combinación de dos tipos diferentes de electrodos para la transferencia génica simple y conveniente en las zonas objetivo de desarrollo del cerebro. En primer lugar, la celebración de un método único de embriones mediante un cable de fibra óptica de luz fría hará que los embriones pequeños (a partir de E9.5) visible de inyección dirigida solución de ADN en los ventrículos y el tipo de aguja electrodos de inserción para el área del cerebro dirigido 5,6. El patrón del cerebro tales como el área cortical se producen en la etapa embrionaria, por lo tanto, estos principios de la electroporación de E9.5 hacer una gran contribución para comprender los patrones de eventos toda la zona. En segundo lugar, la forma precisa de un capilar previene el daño del útero, haciendo agujeros mediante la inserción de los capilares. Además, la forma precisa de los electrodos de aguja se crean con el tungsteno y el alambre de platino y afilado con papel de lija y aislados con esmalte de uñas 7, un método que se describe en detalle en este protocolo. Esta técnica única permite la transfección de ADN plásmido en las áreas restringidas del cerebro y permitirá a los embriones pequeños para ser electroporación. Esto ayudará a, abra una ventana nueva para muchos científicos que están trabajando en la diferenciación celular, la migración celular, para guía de axones en fase embrionaria muy temprana. Además, esta técnica permitirá a los científicos para transfectar ADN plásmido en las partes profundas del cerebro en desarrollo, como el tálamo y el hipotálamo, donde muchos no específicos de la región las líneas de CRE existen para ganancia de función (GOF) o pérdida de la función (LOF), analiza.

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Un método de transferencia de genes en el cerebro del ratón en desarrollo se describe mediante el uso de un método único quirúrgica y la forma especial de los electrodos. Esta técnica única permite la transfección de ADN de plásmido temporal y espacialmente, lo que ayudará a muchos neurocientíficos en el estudio de desarrollo del cerebro.

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.8K vistas.  RIKEN Brain Science Institute. Un método de transferencia de genes en el cerebro del ratón en desarrollo se describe mediante el uso de un método único quirúrgica y la forma especial de los electrodos. Esta técnica única permite la transfección de ADN de plásmido temporal y espacialmente, lo que ayudará a muchos neurocientíficos en el estudio de desarrollo del cerebro.

Video: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

En el útero de electroporación seguido por cultivos neuronales primarias para estudiar la función génica en el subconjunto de neuronas corticales

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Entrega génica eficaz en los territorios del SNC múltiples utilizando In Utero La electroporación

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Generación de ratas transgénicas por vía tópica<em&gt; En el útero</em&gt; La electroporación y<em&gt; En vivo</em&gt; Detección bioluminiscencia

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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 ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Específica Striatum Cortex, hipocampo, Thalamus-, hipotálamo, núcleo-septal lateral y<em&gt; In Utero</em&gt; Electroporación en el ratón C57BL / 6

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Time-lapse Imágenes confocales de las neuronas migratorias en la cultura organotípica en rebanadas de cerebro de ratón embrionario<em&gt; En Utero</em&gt; Electroporación

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Inducción de la recombinación del Cre-lox en la corteza Cerebral de ratón a través de electroporación en el útero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...