Los autores desean agradecer a la tripulación de Stephen dysfusion cDNA de anticuerpos, Dis y moscas w 1118.

1. De recogida de embriones <ol><li> Establecer las jaulas a 25 ° C con 2-4 días de edad, w 1118 placas de jugo de flies.Grape se cambian cada hora durante el día para sincronizar la recolección de huevos en periodo de 1-2 días antes de la recolección </li><li> Recoger los embriones durante 1 hora a 25 ° C </li><li> Corte un pedazo rectangular de agar jugo de uva, de corte ligeramente en la mitad con una cuchilla de afeitar, dejar una línea en el agar </li><li> Utilizar una sonda de metal para la transferencia de embriones a partir de la placa de jugo de uva con su trozo de agar jugo de uva, que se alinean en una línea recta con el eje más largo del embrión en un ángulo de 45 grados a la línea en el centro del agar. Obtener una línea recta de unos 50 embriones. Asegúrese de que todos los punto de embriones en la misma dirección, con el extremo posterior en dirección opuesta a la línea. </li><li> Corte un pedazo rectangular de cinta adhesiva doble, y el lugar a un cubreobjetos 18x18mm. </li><li> Recoger los embriones con cinta adhesiva doble en la hoja de la cubierta </li><li> Coloque la hoja de la cubierta de una lámina de vidrio y seco del embrión en el aire durante unos 10 min. </li><li> Cubrir el embrión de una fina capa de aceite de halocarbonos 700, ahora está listo para la inyección. </li></ol> 2. Tirar la aguja Hacen que las agujas de microinyección tirando tubos capilares. Nosotros usamos un extractor de aguja de precisión de instrumentos del mundo (modelo PUL-1). Las especificaciones extractor aguja programa son: calor: clase 1, el retraso 4. 3. Llenado de las agujas La carga de back-agujas con la pipeta Eppendorf P20 equipada con puntas Eppendorf Microloader, normalmente la carga 1.5-2 dsRNA l 4. Quiebre de las agujas <ol><li> Antes de romper la aguja, coloque un cubreobjetos sobre un portaobjetos. </li><li> Prepara un portaobjetos con una gota de aceite en el centro de la diapositiva </li><li> Encienda el picopump Picospritzer III para ajustar la presión del aire </li><li> Romper la aguja llena hasta el borde de una hoja de cubierta, creando una punta afilada. </li><li> Cuando la punta se rompe, el paso en el pedal de Picospritzer III picopump, algunos dsRNA se saldrá de la punta de la aguja. </li><li> Sacar el portaobjetos con cubreobjetos. </li><li> Inserte la aguja debajo de la gota de aceite en el centro de la diapositiva preparada antes. </li><li> Ajustar las configuraciones en el picopump Picospritzer III para obtener el 100-200PL dsRNA cuando usted pisa el pedal. dsRNA puede ser visto como una pequeña gota. </li></ol> 5. Inyección <ol><li> Llevar la punta de la aguja a la parte posterior del embrión blastodermo primera vez en el mismo plano focal y se inyecta el embrión fuera del centro alrededor del 30-50% la longitud de huevo. </li><li> Después de pisar el pedal del picopump Picospritzer III, una pequeña cantidad de dsRNA aparecerá como un punto pequeño en el sitio de la inyección. </li></ol> 6. El análisis fenotípico <ol><li> Después de la inyección, colocar la placa en una cámara húmeda cubierta (placa Petri de plástico) a 18 ° C hasta que los embriones se desarrollan en la etapa embrionaria deseado. </li><li> Use la sonda para eliminar los embriones de la cinta de doble cara y empujarlos al borde de la hoja de la cubierta. Lavado de los embriones de la diapositiva en un frasco de colección con una malla de nylon en la parte inferior con heptano, lavar 3 veces con PBT. Embriones Dechorionate en el 50% de lejía durante 5 minutos, y lavar 3 veces con PBT. La transferencia de embriones a partir de la colección vial a un pedazo de malla de nylon, y luego sumergir la malla de nylon en la solución de fijación (5 ml de heptano, 4.5ml de PBS, 0,5 ml de formaldehído 37%) para liberar los embriones. Fix embriones en la solución fijadora durante 30 minutos. Devitellinize embriones con metanol, la transferencia de embriones en un tubo eppendorf y inmunotinción los embriones a través de protocolos estándar. </li><li> Los embriones también pueden desarrollarse a la etapa larval adecuado. </li><li> Preparar un portaobjetos con una gota de aceite de halocarbonos 700 en el centro </li><li> Transferencia de una larva a la diapositiva, ponga una hoja de cubierta en la parte superior </li><li> Observar las larvas bajo el microscopio de campo claro. </li></ol> 7. Los resultados representativos: Un ejemplo de dsRNA derribar dysfusion (días) de genes en embriones Drosophlia se muestra en la Fig. 1. GFP-dsRNA embriones inyectados son el control negativo. Disfunción es un factor de transcripción bHLH PAS expresa en las células de fusión traqueal. GFP-dsRNA embriones inyectados mostrar fusión traqueal normal, y la proteína Dis está presente en las células de fusión (Figura 1). Por otro lado, la dis-dsRNA inyecta embriones muestran la fusión no poder, y las proteínas Dis no están presentes en las células de fusión (fig. 1B). Cuando se inyecta dsRNA embriones se desarrollan en estado larvario, las larvas disfunción dsRNA inyectados mostraron la fusión no poder (Fig.1D) en comparación con las buenas prácticas agrarias dsRNA control inyectados negativo (fig. 1C). Estos resultados mostraron eficaces tumbar de la expresión endógena del gen disfuncional por dsRNA inyección en embriones de Drosophila <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="Figura 1" /> Figura 1. Eliminación de la función de disfunción de la dis-RNAi revela traqueal fusión defects. Embriones blastodermo (w 1118) fueron inyectados con las buenas prácticas agrarias dsRNA (control negativo) o disfunción dsRNA y se analizaron para detectar defectos traqueal. (A) Fase 16 embriones inyectados con las buenas prácticas agrarias dsRNA y teñidas con α-Dis y MAb 2A12 que las manchas de la luz traqueal. Prominente que se muestra es el lateral del tronco, que se ha fundido. Las flechas apuntan a las células troncales de fusión lateral dis-positivo. (B) Etapa 16 embriones inyectados con disfunción dsRNA y teñidas con α-Dis y 2A12 MAb. El embrión mostró una falta de fusión lateral del tronco (el asterisco indica la ubicación normal de una fusión lateral del tronco en los embriones de control). No se observan células dis-positivos fueron observados, lo que indica que la dis-ARN supresión efectiva de proteínas Dis. (C y D) En segundo lugar, instar las larvas, ya sea con las buenas prácticas agrarias dsRNA o disfunción dsRNA fueron examinadas por microscopía de campo brillante para los defectos traqueal. (C) Sitios de la fusión se señalan con un asterisco en las buenas prácticas agrarias dsRNA larvas de control de inyección. (D) El lateral del tronco no se funden en las larvas de la dis-ARN inyectada, (*) indica la posición normal de una fusión lateral del tronco.

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, 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=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

La inyección de dsRNA presente método permite aquí un análisis muy sensible y rápida de la función de genes en el desarrollo de Drosophila traqueal. Este método potencialmente puede ser aplicado para analizar la función de genes de otros tejidos y el desarrollo de órganos.

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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
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<td>Picospritzer III picopump</td>
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<td>Parker Precision Fluidics</td>
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rnai interference by dsrna injection into drosophila embryos

Una prueba genética es uno de los métodos más eficaces para obtener conocimientos en un proceso biológico complejo. Con los años muchas mejoras y herramientas para la manipulación genética se han convertido en disponibles en Drosophila 2. Poco después del descubrimiento inicial de Frie y Mello 3 ARN de doble cadena que puede ser utilizado para derribar la actividad de genes individuales en Caenorhabditis elegans, el ARN de interferencia (ARNi) ha demostrado proporcionar una poderosa aproximación genética inversa para analizar las funciones de genes en el desarrollo de Drosophila órgano 4, 5. Muchos órganos, incluidos los de pulmón, riñón, hígado y sistema vascular, se componen de redes ramificadas tubular que transportan líquidos o gases vitales 6, 7. El análisis de la formación de Drosophila traqueal proporciona un excelente sistema modelo para estudiar la morfogénesis de otros órganos tubulares 8. El Berkeley Drosophila Genome Project ha revelado cientos de genes que se expresan en el sistema traqueal. Para estudiar el mecanismo molecular y celular de la formación del tubo, el reto es entender el papel de estos genes en el desarrollo de la tráquea. En este sentido, se describe un método detallado de dsRNA inyección en embriones de Drosophila para la expresión de genes individuales caída. Con éxito derribado dysfusion endógena (días) la expresión génica por inyección de dsRNA. Disfunción es una proteína bHLH-PAS expresa en las células de fusión traqueal, y se requiere para la rama traqueal fusión 9, 10. disfunción RNAi elimina completamente la expresión disfunción y dio lugar a defectos de fusión traqueal. Este método relativamente simple proporciona una herramienta para identificar los genes requried para tissure y desarrollo de los órganos de la Drosophila.

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

ARN de interferencia se ha demostrado muy eficaz para analizar la función del gen en<em&gt; Drosophila</em&gt; El desarrollo traqueal. Un protocolo detallado utilizado por Jiang laboratorio para inyectar dsRNA en embriones de la mosca de la expresión génica caída se ilustra. Esta técnica tiene el potencial de genes de detección necesarios para el desarrollo de órganos y tejidos en<em&gt; Drosophila</em&gt;.

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 ...

rnai-interference-by-dsrna-injection-into-drosophila-embryos

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K vistas.  Oakland University. ARN de interferencia se ha demostrado muy eficaz para analizar la función del gen en Drosophila El desarrollo traqueal. Un protocolo detallado utilizado por Jiang laboratorio para inyectar dsRNA en embriones de la mosca de la expresión génica caída se ilustra. Esta técnica tiene el potencial de genes de detección necesarios para el desarrollo de órganos y tejidos en Drosophila.

Video: RNAi Interference by dsRNA Injection into Drosophila Embryos

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

La inyección de dsRNA en Mujer A. aegypti mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Investigación

Biología

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

La disección en vivo de Drosophila Los embriones: métodos simplificados para la detección colecciones mutantes tinción con anticuerpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

En vivo-La centrifugación de embriones de Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Entrega bacteriana de RNAi efectores: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagen de la posición vertical Drosophila Los embriones

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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 ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi gatillo en la entrega<em&gt; Anopheles gambiae</em&gt; Las pupas

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.