Este trabajo fue apoyado por becas de la Fundación de Investigación de la Sordera Yc S y la NSF (IOS 0930096) con el KFB. KFB: NIH / 2 NINDCD RO1 DC04184, <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184-08W1</a> , NSF DBI 0832862, NSF IOS 0930096

1. Electrodos para la toma de electroporación <ol><li> Cortar 75 m de diámetro de alambre de tungsteno (AM Systems, Inc. Carlsborg, WA) de longitud adecuada (alrededor de 5.3 cm) </li><li> Poner alambre de tungsteno a través de cable conector molex (latón estampado pin) </li><li> Envuelva los cables de tungsteno y el conector del cable molex con tubo retráctil (SPC Technology, Chicago, IL) y aplicar calor con quemador Bunsen o una pistola de calor para sellar el tubo de la reducción al cable </li><li> Para afilar la punta del alambre de tungsteno, sumergir el alambre en hidróxido de sodio 1,0 N (Conrad et al. 1993) y, con un clip en el otro electrodo, electrolizar el alambre con un Electroporador onda cuadrada. Las condiciones que utilizamos son 5 de 50 voltios con una duración de impulsos de 100 ms con 50 ms entre pulsos. Los electrodos deben ser afilados con un diámetro de 15-20 micras </li><li> Mantenga los cables en una bandeja en las ranuras presionado en arcilla antes de su uso en la electroporación </li></ol> 2. Configurar la estación de electroporación <ol><li> Cinta afilada 75μm electrodos de tungsteno para micromanipulador (instrumentos de precisión Mundial). </li><li> Micromanipuladores lugar a ambos lados de la platina del microscopio de disección (Leica). </li><li> Conectar los electrodos a una Proteger CUY-21 Electroporador Edición de onda cuadrada (Figura 1). </li><li> Encienda el electroporador y establecer parámetros para la electroporación, como el voltaje, la duración del pulso, y el número de pulsos. Los parámetros más eficaces para este experimento se encontró que tres de 13 voltios de pulsos que se prolongó durante cada 5.0ms, con 100 ms entre cada pulso. Si electroporación crea burbujas de aire en el embrión, reducir la duración del pulso. </li></ol> 3. Microinyección de Morpholinos Dentro vesícula ótica pez cebra <ol><li> Embriones de pez cebra de la cosecha de un estanque de cría. Incubar los embriones en el embrión de conciencia medio con 0,3 ppm de azul de metileno (Westerfield, 2005) a 28,5 ° C durante la noche. </li><li> Tire las agujas de cristal con un extractor de Sutter P-97 electrodos </li><li> Calentar una base de gel de agarosa (agarosa al 1% en los peces de agua en un 10 mm placa de Petri) a 37 ° C en un baño de agua </li><li> Calentar 1% de agarosa de bajo punto de fusión (LMPA) en peces de agua hasta que se derrita y mantenerlo caliente en el baño de agua a 37 ° C. </li><li> Llene una aguja de vidrio con una solución de morfolino (GeneTools, Corvallis, Oregón) y cortar la punta de diámetro apropiado. Monte la aguja de inyección en un micromanipulador (Kuhn). </li><li> Cortar punta de la aguja de aproximadamente 10 micras y se inyecta en aceite mineral para asegurarse de que la punta permite la entrega suficiente de solución de morfolino. </li><li> Dechorinate embriones a las 24 horas postfertilización (HPF) y anestesia con MS222 (tricaína, Sigma) en agua 0,3 PPM pescado azul de metileno. </li><li> Alinear 3 a 5 embriones anestesiado en la base de gel de agarosa con el lado derecho para el análisis uniforme conveniente </li><li> Ponga 1 a 2 gotas de 1% sobre LMPA cada embrión y dejar solidificar a fijar el embrión en su lugar </li><li> Gire la placa de Petri para que la vesícula ótica se encuentra en el lado derecho </li><li> Coloque la aguja en el lumen de la vesícula ótica e inyectar la solución morfolino con un neumático PicoPump PV820 (Instrumentos del Mundo de Precisión) con un tanque de nitrógeno unido (Figura 2) </li></ol> 4. Electroporación procedimiento <ol><li> Mueva los embriones montado en la estación de electroporación inmediatamente después de la inyección </li><li> Coloque el electrodo positivo en el tejido que está por detrás de la vesícula ótica, pero no penetran, insertar el electrodo negativo en el cerebro, justo por delante de la vesícula ótica </li><li> Aplicar actual mediante un pedal o pulsar un interruptor. </li><li> Vierta el agua los peces de agarosa y eliminar los embriones de agarosa con remolinos y una pipeta de vidrio. Tenga cuidado de no dañar los embriones. Para los embriones que son difíciles de eliminar, el uso de una aguja para romper con suavidad a la basura cualquier LMPA que rodea al embrión. </li><li> Embriones a cabo en un plato con pescado de agua azul de metileno y elevar los embriones a los escenarios deseados para el análisis (morfológico, inmunohistoquímico, en anlaysis hibridación in situ, etc y microscópicas). </li></ol> 5. Los resultados representativos: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Figura 1" /> Figura 1. Estación de electroporación. Los electrodos se realiza mediante cables de tungsteno afilada 75μm y conectados por cables a una Proteger CUY-21 Electroporador onda Editar Square. Estos electrodos fueron grabadas a micromanipulador. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Figura 2" /> Figura 2. Estación de inyección. Todos los embriones fueron inyectados en el oído derecho, para fines de normalización. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Figura 3" /> Figura 3. Faloidina tinción de los filamentos de actina con 3 embriones fdd. (A) El embrión 3 fdd injected con el control de MO tiene una fuerte tinción de phalloidin en la mácula posterior (pm). (B) El embrión que fue inyectado con el control de MO y luego electroporated tenían niveles similares de tinción phalloidin en la tarde en los embriones de inyección única. (C) Inyección de la OM del FOMIN en la vesícula ótica por sí sola no causa la reducción de la tinción phalloidin, mientras que la inyección de MOs FOMIN junto con la electroporación causado una drástica reducción de la tinción phalloidin en la tarde (D). am, mácula anterior. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Figura 4" /> Figura 4. Manchas faloidina de filamentos de actina con larvas DPF 5. No hubo diferencias entre la inyección única (A) y la inyección con la electroporación, cuando la norma morfolino de control se utilizó (B). Sin embargo, las larvas DPF 5 que fueron inyectados con mif morfolinos y electroporated demostraron una reducción de tinción phalloidin en la mácula posterior (pm) (D), aunque menos pronunciado que tres fdd, cuando se compara con el embrión que fue inyectado con mif morfolinos sólo (C ). am, mácula anterior. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Figura 5" /> Figura 5. Tinción tubulina acetilada de los embriones a los 3 fdd. El embrión en (A) no recibió ninguna inyección o electroporación. El embrión en (B) se electroporated pero no recibió ninguna inyección. Los embriones que reciben tanto de la inyección y la electroporación (C, D) con mif morfolinos había disminuido visiblemente manchas tubulina en comparación con los controles. (Por la tarde, la mácula posterior). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Figura 6" /> Figura 6. Tinción tubulina acetilada de los embriones a los 3 fdd con el control de morfolino. (A) de embriones sin ningún tipo de tratamiento mostraron una fuerte tinción tubulina acetilada en la mácula posterior (pm) y la inervación extensa. (B) de embriones inyectados con el control de morfolino. (C) de embriones tratados con la electroporación solamente. (D) de embriones de control con el control de morfolino inyectado y electroporated tiene niveles similares de tubulina acetilada en la mácula y la inervación posterior comparación con el control sin tratamiento.

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

La producción de este artículo fue patrocinado por Gene Herramientas, LLC, que producen los reactivos mencionados en el video y el texto.

Hemos introducido con éxito MOs oligonucleótidos antisentido en el oído del embrión de pez cebra 24 HPF. Los embriones que se plantearon después de la inyección y la electroporación fueron viables. Si los embriones sobrevivieron a la electroporación, que creció a un ritmo normal, comparado con los embriones que no habían recibido tratamiento, así como los embriones que sólo había sido inyectado y embriones que habían sido sólo electroporación. Hemos observado los efectos de la MIF y MIF-OM como el oído después de la inyección y la electroporación mediante el etiquetado con phalloidin y tubulina acetilada. Tinción phalloidin de filamentos de actina en las células ciliadas de la mácula posterior indica que la electroporación de MIF y MIF-MO como en la etapa 24 HPF causado cambios en la célula de pelo y / o números de estereocilios en comparación con los embriones que fueron inyectados sólo con el OM (Figura 3). Esta disminución en el número de células de cabello es consistente con los hallazgos de Shen et al. (Presentado) que MOs para MIF y MIF-como causa una reducción en el número de células ciliadas de la mácula sacular. La disminución del número de células de cabello en los embriones que fueron inyectados con OM y electroporated demostrar que la electroporación puede ayudar a mover el OM a través de la membrana celular, lo que aumenta el alcance de los efectos morfológicos en comparación con los embriones en los que se inyectó la vesícula ótica solamente. Los cambios en la morfología del ganglio statoacoustic se observaron mediante la tinción tubulina acetilada. El grado de inervación por el ganglio statoacoustic se vio afectada, ya que los embriones que fueron inyectados y electroporated muestran una disminución en la ramificación de las neuritas (Figura 5). También puede haber disminución de la condensación de las células ganglionares y / o disminución del número de células ganglionares, ya que la tinción tubulina acetilada se redujo significativamente en los embriones que fueron inyectados y electroporación. Debido a que el FOMIN ha demostrado ser fundamental para el desarrollo del sistema nervioso (Suzuki et al., 2004), los cambios observados después de la electroporación puede ser el resultado de la transfección aumento de la MIF y MIF-MO como solución a las células neuroblast. Electroporación de antisentido oligo morfolinos directamente en un tejido en desarrollo es una herramienta útil para controlar la expresión de un gen durante el desarrollo de ese tejido, tanto espacial como temporalmente, en el embrión. Electroporación de MIF y MIF-como Mos en los tejidos del oído interno como resultado de la morfología anormal de la mácula tanto posterior y el ganglio statoacoustic. Hubo una reducción en el número de células ciliadas de la mácula posterior de los embriones que habían sido inyectados y electroporación en comparación con los embriones que habían recibido ningún tratamiento o con los embriones que habían sido inyectados o sólo electroporated solamente. También hubo una disminución en el grado de inervación de la revisión posterior por el ganglio sensorial statoacoustic y el tamaño de la SAG. La electroporación es un método valioso para la transfección de las macromoléculas en tejidos específicos, con la ventaja de observar los efectos primarios de ese particular de ADN, el ARN, o MO en el tejido deseado y la discriminación de estos efectos secundarios en otros tejidos, incluyendo el cerebro, la cresta neural y mesénquima periotic en el desarrollo del oído interno (Barald y Kelley, 2004).

<ol><li> OM: 0,3 mM de una combinación de la MIF, FOMIN-como MO1, y el FOMIN-como MO2 se utilizó. Mif MO es gratuito para el codón de inicio de pez cebra de mRNA del FOMIN: 5&#39;-acatcggcatgactgcgacagagat-3 &#39;. Dos MOs fueron utilizados para el FOMIN como mif-como MO1 es gratuito en el sitio de inicio de traducción:. 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;, mientras que el FOMIN como MO2 derivado de que la secuencia en la intron1/exon dos límites: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC -3 &#39;. Control de MO fue la secuencia de nucleótidos de Herramientas revueltos Gene: 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;. </li><li> azul de metileno (0,3 ppm) en los peces de agua </li><li> MS222 (tricaína, Sigma; 0,64 mm en los peces de agua) </li><li> Bajo punto de fusión de agarosa (LMPA; Roche) </li><li> rojo fenol (Sigma) </li><li> Anti-tubulina acetilada (Sigma) </li><li> Alexa Fluor ® 488 cabra anti-ratón segundo anticuerpo (sondas moleculares) </li><li> Texas rojo-phalloidin (sondas moleculares) </li></ol>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

En los últimos años, la electroporación se ha convertido en una técnica popular para la transfección in vivo del ADN, el ARN y morfolinos en varios tejidos, incluyendo el ojo, el cerebro y somitas de pez cebra. La ventaja de la electroporación sobre otros métodos de manipulación genética es que los tejidos específicos puede ser objetivo, tanto espacial como temporalmente, para la introducción de las macromoléculas de la aplicación de corriente eléctrica. A continuación se describe el uso de la electroporación para transfectar mif y morfolinos mif-como en los tejidos del oído interno en desarrollo del pez cebra. En estudios anteriores, el FOMIN morfolino se inyectan en embriones en el 1 - al estadio de 8 células resultado en general los cambios morfológicos en el sistema nervioso y los ojos, así como el oído. Al dirigirse a los tejidos del oído interno en las etapas posteriores del desarrollo, podemos determinar los efectos primarios del FOMIN en el oído interno en desarrollo, en comparación con los efectos secundarios que pueden resultar de la influencia de otros tejidos. Mediante el uso de tubulina acetilada y phalloidin tinción para estudiar la morfología de las neuronas, los procesos neuronales y las células del cabello asociada con la mácula posterior, hemos sido capaces de evaluar la eficacia de electroporación como método de transfección dirigida en el oído interno del pez cebra. Las vesículas óticas de los embriones fueron inyectados con 24hpf morfolinos y electroporación y fueron comparados con los embriones que habían recibido ningún tratamiento o sólo se había inyectado o electroporación. Los embriones que fueron inyectados y electroporación mostró una disminución en el número de células de cabello, disminución de la inervación del ganglio statoacoustic (SAG) y menos neuronas SAG en comparación con los grupos control. Nuestros resultados mostraron que la entrega directa de morfolinos en otocysts en etapas posteriores se evita el sistema nervioso no específica y los efectos de la cresta neural de morfolinos en las fases celulares 1-8. También permite el examen de los efectos que se dirigen hacia el oído interno y no los efectos secundarios en el oído de los principales efectos en el cerebro, mesénquima de la cresta neural o periotic.

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Un método para entregar morfolinos directamente en el pez cebra en otocyst 24hpf se ha desarrollado. Con microinyección de morfolinos en el lumen de la vesícula ótica y la electroporación para llevar a cabo la penetración, hemos sido capaces de evitar el efecto de morfolinos en el cerebro y obtener efectos específicos en el oído interno.

Entrega directa de Morpholinos FOMIN Hacia el otocyst pez cebra por inyección y electroporación afecta el desarrollo del oído interno

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

Research

JoVE Journal

Biology

14.2K vistas.  University of Wisconsin, Madison. Un método para entregar morfolinos directamente en el pez cebra en otocyst 24hpf se ha desarrollado. Con microinyección de morfolinos en el lumen de la vesícula ótica y la electroporación para llevar a cabo la penetración, hemos sido capaces de evitar el efecto de morfolinos en el cerebro y obtener efectos específicos en el oído interno.

Video: Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

La disección fina y gruesa de los epitelios sensoriales del oído interno en adultos de pez cebra ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Investigación

Biología

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Inyección intraperitoneal en adultos de pez cebra

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi interferencia por inyección de dsRNA en Drosophila Los embriones

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporación de mesénquima craneofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En electroporación in vivo de Morpholinos en la regeneración de adultos de pez cebra Aleta de cola

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferencia de genes en embriones de pollo por los mayores<em&gt; Ex ovo</em&gt; La electroporación

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Estudio comparativo de los métodos de entrega de fármacos dirigidos a la oreja del ratón interno: Bullostomy<em&gt; vs.</em&gt; Inyección transtimpánicos

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Novedoso método de plásmido ADN entrega al urotelio de la vejiga de ratón por electroporación

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...