Este trabajo fue financiado por la Fundación Alemana de Investigación (Deutsche Forschungsgemeinschaft).

1. Preparación de ADN y vidrio Micropipetas para Inyección <ol><li> Buenas micropipetas de vidrio de calidad son esenciales para reducir la velocidad inicial alta aborto debido a la pérdida de líquido amniótico. El procedimiento para sacar micropipetas de vidrio ha sido bien documentado 13,18,25. Use 1,2 mm de diámetro capilares tirado en una convencional Sutter P-97 dispositivo con los ajustes P = 500; calor = 300; Tire = 40; Velocidad = 50; Tiempo = 50. Coloque el extractor de 3 mm filamentos &quot;valle&quot; (Sutter Instrument FT330B). Los filamentos de tamaño de 2 mm han dado para nosotros los resultados menos satisfactorios. Por otro lado, biselado de la micropipeta consejos no parece mejorar los resultados para nosotros. </li><li> Disolver purificada, plásmido de ADN libre de endotoxina en PBS (cultivo celular de grado) que contiene 0,1% Fast Green a una concentración final de 1 a 2 g / l. The Green Fast hará que la solución inyectada visible en el ventrículo del cerebro embrionario. </li><li> Cargar 10 l de la solución de ADN en la micropipeta de vidrio. </li&gt; <li> Conectar la micropipeta de vidrio para el sistema de inyección (Bomba Pico) o pipeta de boca. </li></ol> 2. Anestesia Prepare la mesa de operaciones con una almohadilla eléctrica y los instrumentos quirúrgicos. Encienda las fuentes de luz fría para facilitar la visualización de los embriones. Desinfectar las herramientas quirúrgicas que utilizan, por ejemplo, un cordón esterilizador de vidrio. Tres procedimientos de anestesia son posibles para este protocolo (ver Discusión). Aquí vamos a describir el que consideramos la mejor, usar la inhalación de isoflurano para la inducción de la anestesia (tasa de flujo de 0,5 L / min), así como de mantenimiento (tasa de flujo de 1 l / min). <ol><li> Introducir el ratón embarazada en un pequeño transparente (por lo que el ratón permanece visible) recipiente conectado a la Komesaroff Marcos 5 Anestésico dispositivo por un tramo corto de tubo. </li><li> Llene el vaporizador con isoflurano, a continuación, abra la botella de oxígeno conectado al dispositivo. Sopla una o dos pulsos de isoflUrane / oxígeno (0,5 L / min) en el recipiente que contiene el ratón y el reloj de la narcosis de establecer pulg </li><li> Tome el ratón fuera, cubrir sus ojos con un ungüento para evitar que se sequen y se ajustan a la máscara de anestesia de flujo en la cabeza de inmediato. </li></ol> 3. Laparotomía <ol><li> Coloque el ratón panza arriba sobre el cojín eléctrico (de lo contrario su temperatura corporal bajará muy rápido, disminuyendo las posibilidades de recuperación) y asegurar su cuerpo en su lugar, fijando sus cuatro patas a los lados con cinta adhesiva. Evaluar la profundidad de la anestesia mediante la comprobación de la pérdida de la respuesta a la estimulación refleja (por ejemplo, los pies o el pellizco de la cola con una presión firme). </li><li> Afeitarse la superficie abdominal y desinfectar con el iodophorpovidone-yodo (Braunoderm). </li><li> Hacer una incisión longitudinal (1 a 1,5 cm de largo) sobre la piel abdominal. Luego se corta el peritoneo. Coloque una gasa de algodón alrededor de la incisión. Hacer un cuerno uterino visible y tire de ella con cuidado con unas pinzas romas en la PBS-enjuagado gauze. Enjuague el útero con PBS muy a menudo para mantener siempre humedecido (Figuras 2A y 2B). Durante todo el procedimiento de evitar tirar la mesometrio o el útero apretado, ya que la presión dentro del útero transmitirá al embrión aumenta las posibilidades de pérdida de fluido de la inyección resulta en aborto. </li></ol> 4. Inyección de ADN en el ventrículo cerebral <ol><li> Mantenga el útero de tal manera que los ventrículos del cerebro pueden ser visualizados. No cambiar la posición ampliamente un embrión que se encuentra en una posición desfavorable - esto sólo aumenta las posibilidades de aborto. </li><li> En cuanto a la cabeza del embrión a partir de la parte superior, localizar visualmente el hueco o fisura entre los hemisferios corticales izquierdo y derecho. Los hemisferios son fáciles de distinguir y los ventrículos laterales dentro de ellos (por no ser objetivo) por lo general pueden ser percibidas como formas un tanto oscuras. Si se encuentra un embrión a ser perfectamente orientada para la inyección de ADN (FigurES 2C y 2D), es posible perforar la pared uterina y entran en el tercer ventrículo a la vez. Mantenga la micropipeta de vidrio a 45 ° a la pared uterina y la punción que en el extremo rostral de la brecha entre los hemisferios corticales, penetrando por alrededor de 1 mm. De esta manera, la punta de la micropipeta de vidrio entrará en el tercer ventrículo del cerebro (no el ventrículo lateral) (Figuras 2C y 2D). En embriones de menos orientados favorablemente es útil primero perforar la pared uterina (siempre a 45 °) en la proximidad de la cabeza embrionaria, colocar la punta de la micropipeta en la posición correcta entre los hemisferios corticales y sólo entonces perforar el cerebro. Inyectar aproximadamente 1 l de solución de ADN en el tercer ventrículo (una buena inyección llena el ventrículo con el líquido verde). Repita el mismo procedimiento con todos los embriones de un cuerno uterino. Esto permite algún tiempo para que la solución de ADN para mezclar uniformemente con el fluido del ventrículo y llegar a la entirneuroepitelio correo. </li></ol> 5. Orientación por el hipotálamo para electroporación <ol><li> Cambie el electroporador y ajuste la configuración según la edad embrionaria (por E12.5 usamos 5 pulsos de onda cuadrada, 50 V, 50 ms ON/950 mseg OFF). Uso como polo positivo del electrodo de aguja de acero inoxidable (CUY550-10) y como polo negativo de un electrodo plano y redondo (CUY700P4L). (Es importante que los electrodos son más pequeños que el embrión, ya que de lo contrario la corriente sólo fluye alrededor del embrión, pero no a través de él.) </li><li> Seleccionar para electroporación esos embriones cuya parte dorsal es hacia arriba (es decir, se volvió hacia el experimentador) (ya que la mayoría de ellos lo son), y descartar aquellos cuya orientación no es favorable. Ahora es seguro tocar al útero con los dedos etanol desinfectados o con guantes. Sosteniendo el útero con fórceps, sin embargo, podría interferir con el flujo de corriente durante la electroporación. </li><li> Pierce la pared uterina por la cabeza del embrión entre el amniotic saco y la placenta empujando hacia abajo a través de él con la punta del electrodo de aguja. Utilice el dedo índice de la otra mano, como bloque de empuje. Acerca de 5 mm de la punta del electrodo deben ser ahora entre el saco amniótico y la pared uterina, a aproximadamente el nivel del mesencéfalo (Figuras 2E y 2F). Recuerde que este es el &quot;electrodo de orientación&quot;, por lo que su posición va a determinar qué es lo más probable que se transfectaron parte del neuroepitelio hipotálamo. El embrión tiene que ser muy suavemente &quot;exprimido&quot; entre los electrodos. </li><li> Con la otra mano, la posición del electrodo plano y redondo fuera de la pared del útero en el lado opuesto de la cabeza del embrión (Figuras 2E y 2G). </li><li> Utilice el interruptor de pedal para aplicar tensión (50 V, 50 mseg ON, 950 mseg OFF, 5 pulsos de onda cuadrada). </li><li> Tire lentamente el electrodo de aguja del útero mientras que la celebración posterior del útero con el dedo índice de la otra mano - si amniolíquido tic se pierde a través de la pared perforada, por lo general el embrión será sometido a aborto. Repita el procedimiento con todos los embriones. </li><li> Devuelva el cuerno uterino en el abdomen y repetir la inyección y la electroporación en el cuerno remanente. </li></ol> 6. Para terminar la operación <ol><li> Después de la inyección y la electroporación de todos los embriones, colocar el útero en el abdomen con mucho cuidado, posicionada exactamente como eran antes y húmeda de la cavidad peritoneal con solución salina antes de cerrarlo. </li><li> Suturar el peritoneo con catgut quirúrgico (Vicryl Polyglactin 910, 5-0, Ethicon V4914). Para el cierre de la utilización piel &quot;interrumpió la puntada&quot; methodwith una sutura más resistentes (Supramid Nylon, 6-0, Serag Wiessner A 07171L). </li><li> Desinfectar la superficie del abdomen con povidona yodada (Braunoderm) e inyectar un no-esteroides anti-inflamatorios por vía subcutánea (por ejemplo, 100 l de una solución de 1:10 de Rimadyl en NaCl al 0,9%) o, mejor aún, un opioide como buprenorphine (Temgesic, 0,05 a 0,1 mg / kg de peso corporal en 0,9% NaCl) para aliviar el dolor. </li><li> Retire la madre de la máquina de anestesia y colocarlo en una jaula que es calentada por una resistencia de calentamiento. Supervisar el ratón continuamente hasta que esté completamente recuperado de la anestesia. Más tarde, visita a los animales diariamente para asegurar que se están recuperando de un procedimiento sin ningún signo de infección o dolor. </li></ol> 7. El análisis de los resultados <ol><li> Cosecha de los embriones (o postnatals) de acuerdo con el día de análisis deseado. Ratones postnatales (1 a 2 días de edad) se sacrificaron por decapitación. </li><li> Separar cada embrión de acuerdo con la anotación electroporación anterior. Diseccionar el cerebro bajo un estereomicroscopio y comprobar bajo el microscopio para la señal fluorescente verde (GFP del reportero) en la región apropiada. </li><li> El cerebro seleccionado puede ser analizada &quot;fresco&quot;: fijar el tejido durante un corto período de tiempo en 4% de paraformaldehído y incrustar en agarosa al 4% o en una gelatina-lbumin, a continuación, la sección en un dispositivo de tipo vibratomo (Figura 3). Para el análisis inmunohistoquímico más detallada (Figura 4), ​​crio-proteger el cerebro en solución de sacarosa al 30%, incrustar en medio de montaje (OCT Tissue Tek) luego se corta secciones 20 micras en un criostato. </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Acerca de la anestesia: Desde electroporación en el útero en el hipotálamo puede ser técnicamente difícil y requerir tiempos más largos narcosis, preferimos para inducir y mantener la anestesia mediante la administración de una mezcla de oxígeno e isoflurano. En nuestra experiencia, los animales se mantienen adecuadamente anestesiado de esta manera por períodos de hasta una hora por lo menos, la recuperación de la madre es muy rápido, y la supervivencia de los embriones mejorar. Otros enfoques para l...

La manipulación genética del cerebro de ratón embrionario es un enfoque preferido para aprender acerca de la regulación del desarrollo. La generación de líneas de ratones mutantes sin embargo, es lento y caro. Un método poderoso para introducir cambios genéticos específicos en el desarrollo de las neuronas del cerebro de mamífero es la electroporación en el útero. Esencialmente, la técnica consiste en transfectar ADN en el neuroepitelio embrionario del cerebro por medio de impulsos eléctricos, a continuación, permitiendo que el embrión para sobrevivir durante un cierto período de tiempo, recoger el cerebro y examinarlas para su posible novela, fenotipos informativos. De esta manera, el experimentador puede probar hipótesis casi de inmediato y sin los largos períodos de espera necesarios para la producción de mutantes de ratón. La transfección de ADN en embriones en desarrollo se inició con la electroporación in ovo en embriones de pollo 1. La prueba de concepto esencial para el ratón se realizó en la cultura <shasta&gt; 2. Esto fue seguido por las primeras descripciones de la técnica en el ratón en el útero 3,4. El principal problema es para transfectar el cerebro en desarrollo de los embriones en el útero sin matarlos o de la madre. Aprender a realizar la cirugía necesaria (laparotomía, inyección, electroporación) requiere un largo periodo de entrenamiento. Una vez que la cirugía ha sido dominado hasta el punto en que la tasa de supervivencia embrionaria es aceptable, la pregunta clave es: ¿qué estructuras cerebrales son accesibles? No es sorprendente que los primeros trabajos publicados que muestran los resultados obtenidos con la electroporación in utero se centraron en el desarrollo cortical 5-9. Esto sigue siendo cierto para la mayoría de las publicaciones utilizando esta técnica, ya que la región del cerebro de ratón en desarrollo más accesible a los procedimientos quirúrgicos es la corteza (Figura 1). El procedimiento para la electroporación en el útero en la corteza ha sido descritaen la prensa 10 y 11 a 14 en el vídeo. Una modificación de la técnica se puede utilizar para tratar una parte ventral del telencéfalo, los ganglios basales 15. Más allá del telencéfalo, el diencéfalo (clásicamente dividido en tálamo y el hipotálamo) es una región del cerebro anterior más difícil de alcanzar. Un pequeño número de documentos de informes dirigidos a su parte dorsal y más accesible, el tálamo 16-19. El hipotálamo es la parte más ventral del cerebro anterior, por lo tanto, el uno localizada más profundamente a partir de la superficie dorsal (corteza) (Figura 1). Esta región sigue siendo un reto difícil para los investigadores comprometidos para manipular genéticamente el cerebro del ratón en el útero. Hasta donde sabemos, sólo muy pocos artículos informen sobre los resultados de la transfección útero en el hipotálamo de ratón 20,21. Sin embargo, la importancia funcional del hipotálamo Cañot ser exagerada, ya que regula los comportamientos como comer y beber, el apareamiento, la reproducción y crianza de los hijos 22. Por otra parte, las alteraciones en el desarrollo hipotalámico contribuyen a originar más adelante en las condiciones de vida como la obesidad, la hipertensión, la diabetes y la pubertad precoz 23. Ser capaz de alterar genéticamente el hipotálamo durante el desarrollo sería una herramienta muy poderosa para entenderlo. El protocolo quirúrgico básico para la laparotomía de ratones embarazadas que usamos aquí es similar a la utilizada en otros protocolos 11,13,14,24. Vamos a describir aquí brevemente para completar. Clave para nuestro procedimiento, por otro lado, son del tipo de anestesia, el lugar de la inyección, el tipo de electrodos y la inserción y la posición del electrodo positivo con respecto a la cabeza del embrión. Nosotros preferimos para inducir y mantener la anestesia por inhalación de gas sobre la anestesia intraperitoneal sencilla, ya que el primero permite laperíodos algo más largos de la narcosis necesarios para una cirugía difícil. Resultados inhalación isoflurano en la recuperación más rápida de la anestesia, ya que por lo general la madre demuestra un comportamiento normal ya minutos después de la cirugía. El punto más fácil de la inyección de la solución de ADN con la micropipeta de vidrio es el ventrículo lateral, que sin embargo es completamente inadecuado para la electroporación hipotálamo. Inyección directa en el tercer ventrículo es de hecho fundamental orientar la diencephalic estructuras profundas. Es posible transfectar del hipotálamo o de E12.0 E12.5 con electrodos estándar, off-the-shelf. Hemos encontrado algunos de los electrodos fabricados por Nepa Gene (Chiba, Japón) particularmente adecuado para este propósito. Con nuestro procedimiento se obtiene la transfección de todo el neuroepitelio hipotalámica o, transfección regional parcial dependiendo de la orientación del electrodo. Aquí se demuestra la técnica mediante la transfección del cuerpo mammillary, podría decirse quela más profunda y rebajada de todos los núcleos hipotalámicos. Además, se muestra el análisis histológico detallado de las células transfectadas hacia abajo hasta el nivel celular de la resolución. Una comparación de la electroporación en el útero con otros enfoques para transfectar el cerebro en desarrollo en el útero del ratón se puede encontrar en la sección de Discusión.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

La modificación genética de regiones específicas del cerebro de los mamíferos es el desarrollo de un enfoque experimental muy potente. Sin embargo, la generación de nuevos mutantes de ratón suele ser lento y frustrante. Se ha demostrado que el acceso al cerebro de ratón en desarrollo en el útero con la supervivencia post-operatoria razonable es posible. Sin embargo, los resultados de este procedimiento se han reportado casi exclusivamente para la parte más superficial y de fácil acceso del cerebro en desarrollo, es decir, la corteza. El tálamo, una región más estrecha y más medial, ha resultado más difícil objetivo. Transfección en los núcleos más profunda, especialmente aquellos del hipotálamo, es quizás los resultados más difíciles y por lo tanto, muy pocos han sido reportados. Aquí se demuestra un procedimiento para orientar todo el neuroepitelio hipotalámica o parte de él (regiones hipotalámicas) para la transfección por medio de electroporación. Las claves de nuestro enfoque ya narcosis tiempos, inyección en el third ventrículo, y amable y la colocación de los electrodos apropiados. Además, mostramos los resultados de la focalización y el posterior análisis histológico del núcleo hipotalámico más rebajada, el cuerpo mamilares.

La mayoría de las neuronas del hipotálamo nacen entre E11.5 a E15.2, según el análisis de nacimiento-dating en la rata 26 traducido en el desarrollo de algo más corta ratón 27,28. El pico de la neurogénesis hipotálamo se alcanza a E12.5 29-31. En consecuencia, a la edad de transfección elegido para el presente estudio (E12.5), una gran proporción de neuronas hipotalámicas puede ser etiquetado en cualquier nivel rostro-caudal dado. Análisis en E18....

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

A pesar de la importancia clínica y funcional del hipotálamo,<em&gt; En el útero</em&gt; Manipulación genética de su desarrollo rara vez se ha intentado. Te mostramos un procedimiento detallado para<em&gt; En el útero</em&gt; Electroporación en el hipotálamo del ratón y muestran resultados representativos de transfección hipotálamo total y parcial (regional).

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 ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K vistas.  University of Heidelberg. A pesar de la importancia clínica y funcional del hipotálamo, En el útero Manipulación genética de su desarrollo rara vez se ha intentado. Te mostramos un procedimiento detallado para En el útero Electroporación en el hipotálamo del ratón y muestran resultados representativos de transfección hipotálamo total y parcial (regional).

Video: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

Investigación

Neurociencias

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

La electroporación en el útero métodos para estudiar la excitabilidad de las subpoblaciones neuronales y Conectividad Una sola célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirugía estereotáctica para manipulación genética en las células estriatales de cerebro de ratón Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...