Les auteurs tiennent à remercier équipages Stephen dysfusion ADNc, des anticorps Dys et w mouches 1118.

1. Collecte d&#39;embryons <ol><li> Mettre en place des cages à 25 ° C en utilisant 2-4 jours âgé w 1118 plaques de jus flies.Grape sont changés tous les heures pendant la journée pour synchroniser le ramassage des œufs pendant 1-2 jours avant la collecte période </li><li> Recueillir des embryons pour 1h à 25 ° C </li><li> Couper une pièce rectangulaire d&#39;agar jus de raisin, coupe légèrement au milieu avec une lame de rasoir, de laisser une ligne dans la gélose </li><li> Utiliser une sonde métallique de transférer les embryons de la plaque de jus de raisin à votre morceau de agar jus de raisin, les aligner dans une ligne droite avec axe long de l&#39;embryon angle de 45 degrés à la ligne dans le milieu de l&#39;agar. Obtenir une ligne droite d&#39;environ 50 embryons. Assurez-vous que tous les points embryons dans la même direction, avec extrémité postérieure pointant loin de la ligne. </li><li> Couper une pièce rectangulaire de ruban adhésif double, et il place pour une lamelle 18x18mm. </li><li> Ramassez embryons avec du ruban adhésif double bâton sur la lamelle </li><li> Placer la lamelle sur une lame de verre, sèche l&#39;embryon dans l&#39;air pendant environ 10 min. </li><li> Couvrir l&#39;embryon d&#39;une fine couche d&#39;huile de 700 sur les halocarbures, il est maintenant prête pour l&#39;injection. </li></ol> 2. En tirant l&#39;aiguille Faire des aiguilles de microinjection en tirant des tubes capillaires en verre. Nous utilisons un extracteur aiguille de l&#39;instrument de précision du monde (Modèle PUL-1). Les spécifications d&#39;aiguille extracteur programme sont: la chaleur: 1, délai 4. 3. Remplir les Aiguilles Retour à vide en utilisant les aiguilles Eppendorf p20 pipettes munies d&#39;embouts Microloader Eppendorf, normalement la charge de 1,5 à 2 uL ARNdb 4. Cassez les Aiguilles <ol><li> Avant de rompre l&#39;aiguille, placer une lamelle sur une lame. </li><li> Préparer une diapositive avec une goutte d&#39;huile dans le milieu de la diapositive </li><li> Allumez le picopump Picospritzer III pour ajuster la pression de l&#39;air </li><li> Casser l&#39;aiguille remplie contre le bord d&#39;une lamelle, la création d&#39;une pointe acérée. </li><li> Lorsque la pointe est cassée, pas sur la pédale d&#39;Picospritzer III picopump, certains ARNdb aura une fuite hors de la pointe de l&#39;aiguille. </li><li> Sortez la lame avec la lamelle. </li><li> Insérer l&#39;aiguille sous la goutte d&#39;huile dans le milieu de la diapositive préparée avant. </li><li> Réglez le paramètre sur la picopump Picospritzer III pour obtenir 100-200PL ARNdb lorsque vous appuyez sur la pédale. ARNdb peut être vu comme une petite goutte. </li></ol> 5. Injection <ol><li> Apportez la pointe de l&#39;aiguille à la partie postérieure de l&#39;embryon de blastoderme d&#39;abord dans le même plan focal et injecter l&#39;embryon hors du centre de longueur environ 30-50% d&#39;oeuf. </li><li> Après vous faites un pas sur la pédale de l&#39;picopump Picospritzer III, une petite quantité d&#39;ARNdb apparaîtra comme un petit point au site d&#39;injection. </li></ol> 6. Analyse phénotypique <ol><li> Après l&#39;injection, placer la lame dans une chambre humide couverte (une boîte de Pétri en plastique) à 18 ° C jusqu&#39;à ce que les embryons se développent jusqu&#39;au stade désiré embryonnaire. </li><li> Utilisez la sonde pour chasser les embryons de l&#39;adhésif double face et de les pousser vers le bord de la lamelle. Laver les embryons de la lame dans un flacon collection avec filet de nylon à la base en utilisant l&#39;heptane, laver 3 fois avec le PBT. Embryons Dechorionate l&#39;eau de Javel à 50% pendant 5 min, et laver 3 fois avec le PBT. Transfert des embryons à partir du flacon de collecte à un morceau de filet de nylon, puis tremper le filet de nylon dans la solution de fixation (5 ml d&#39;heptane, 4,5 ml de PBS, le formaldéhyde 0.5ml 37%) pour libérer les embryons. Fixer des embryons dans la solution de fixation pour 30min. Devitellinize embryons avec du méthanol, le transfert des embryons dans un tube Eppendorf et immunocoloration des embryons en utilisant des protocoles standard. </li><li> Les embryons peuvent également développer au stade larvaire approprié. </li><li> Préparer une diapositive avec une goutte d&#39;huile sur les halocarbures 700 dans le milieu </li><li> Transférer une larve à la diapositive, mettez une lamelle sur le dessus </li><li> Observez les larves sous microscope en champ clair. </li></ol> 7. Les résultats représentatifs: Un exemple d&#39;ARNdb renverser dysfusion (dys) gène dans l&#39;embryon Drosophlia est montré dans Fig1. GFP-ARNdb embryons injectés sont le contrôle négatif. Dys est un facteur de transcription bHLH PAS exprimée dans les cellules de fusion trachéale. GFP-ARNdb embryons injectés montrent normale de fusion de la trachée et des protéines Dys est présent dans les cellules de fusion (figure 1A). D&#39;autre part, la dys-ARNdb injecté embryons montrent la fusion branche échoué, et les protéines Dys ne sont pas présents dans les cellules de la fusion (figure 1B). Lorsque ARNdb injecté embryons se développent au stade larvaire, les larves dys-ARNdb injecté montré la fusion branche échoué (Fig.1D) par rapport à la GFP-contrôle négatif ARNdb injecté (figure 1C). Ces résultats ont montré efficaces renverser l&#39;expression des gènes endogènes dys par ARNdb injection dans des embryons de drosophile <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="Figure 1" /> Figure 1. Suppression de la fonction dys par dys-RNAi révèle la fusion d trachéaleefects. Embryons de blastoderme (w 1118) ont été injectés soit avec GFP-ARNdb (contrôle négatif) ou dys-ARNdb et analysés pour les défauts trachéale. (A) Etape 16 embryons injectés avec GFP-ARNdb et colorées avec α-Dys et MAb 2A12 que les taches de la lumière trachéale. Bien en vue est le tronc latéral, qui a fusionné. Les flèches indiquent les cellules latérales du tronc de fusion Dys-positif. (B) Étape 16 embryons injectés avec dys-ARNdb et colorées avec α-Dys et MAb 2A12. L&#39;embryon a montré une absence de fusion du tronc latéral (l&#39;astérisque indique l&#39;emplacement normal d&#39;une fusion latérale du tronc chez les embryons de contrôle). Pas de cellules Dys-positives ont été observées, indiquant que la dys-ARN effectivement aboli en protéines Dys. (C à D) Deuxième stade larvaire soit avec GFP-ARNdb ou dys-ARNdb ont été examinés par microscopie à champ lumineux des défauts trachéale. (C) Sites de fusion sont indiqués par un astérisque dans la GFP ARNdb larves de contrôle injecté. (D) Le tronc latéral échoué à fusionner dans les larves dys-ARN injecté, (*) indique l&#39;emplacement normal d&#39;une fusion du tronc latéral.

<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 présente méthode d&#39;injection permet ARNdb ici une analyse très sensible et rapide de la fonction des gènes chez la drosophile développement trachéale. Cette méthode peut potentiellement être appliquée pour analyser la fonction des gènes d&#39;autres tissus et le développement d&#39;organes.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III picopump</td>
<td>&nbsp;</td>
<td>Parker Precision Fluidics</td>
<td>051-0500-900</td>
<td>&nbsp;</td>
<tr>
<td>Micro-pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21170M</td>
<td>&nbsp;</td>
<tr>
<td>Microloaders</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

rnai interference by dsrna injection into drosophila embryos

Le dépistage génétique est l&#39;une des méthodes les plus puissants disponibles pour obtenir un aperçu dans le processus biologique complexe 1. Au fil des ans de nombreuses améliorations et d&#39;outils pour la manipulation génétique sont devenues disponibles chez la drosophile 2. Peu après la découverte initiale par Frie et Mello 3 qui ARN double brin peut être utilisé pour knockdown l&#39;activité des gènes individuels chez Caenorhabditis elegans, l&#39;interférence ARN (ARNi) a été montré pour fournir une puissante approche de génétique inverse afin d&#39;analyser les fonctions des gènes chez la drosophile organes de développement 4, 5. De nombreux organes, notamment du poumon, du rein, le foie et le système vasculaire, sont composés de réseaux ramifiés tubulaire transport de fluides vitaux ou de gaz 6, 7. L&#39;analyse de la drosophile la formation trachéale fournit un excellent modèle pour étudier la morphogenèse des autres organes tubulaires 8. Le Berkeley Drosophila Genome Project a révélé des centaines de gènes qui sont exprimés dans le système de la trachée. Pour étudier le mécanisme moléculaire et cellulaire de la formation du tube, le défi est de comprendre le rôle de ces gènes dans le développement de la trachée. Ici, nous avons décrit une méthode détaillée d&#39;ARNdb injection dans l&#39;embryon de drosophile à l&#39;expression des gènes knockdown individuels. Nous avons réussi à renverser l&#39;expression des gènes endogènes dysfusion (dys) par injection ARNdb. Dys est une protéine bHLH-PAS exprimée dans les cellules trachéales de fusion, et il est nécessaire pour la branche trachéale de fusion 9, 10. dys-RNAi complètement éliminé l&#39;expression dys et a abouti à la fusion anomalie trachéale. Cette méthode relativement simple fournit un outil pour identifier les gènes requried pour tissure et le développement des organes chez la drosophile.

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 interférence a été prouvé très efficace pour analyser la fonction des gènes dans<em&gt; Drosophile</em&gt; Développement trachéale. Un protocole détaillé utilisé par Jiang laboratoire pour injecter des ARNdb dans des embryons voler à l&#39;expression des gènes knockdown est illustré. Cette technique a le potentiel pour le dépistage des gènes nécessaires au développement des tissus et des organes de<em&gt; Drosophile</em&gt;.

Interférences ARNi par injection dans les ARNdb Drosophile Embryons

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 vues.  Oakland University. ARN interférence a été prouvé très efficace pour analyser la fonction des gènes dans Drosophile Développement trachéale. Un protocole détaillé utilisé par Jiang laboratoire pour injecter des ARNdb dans des embryons voler à l'expression des gènes knockdown est illustré. Cette technique a le potentiel pour le dépistage des gènes nécessaires au développement des tissus et des organes de Drosophile.

Vidéo: 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.

L&#39;injection d&#39;ARNdb dans Femme Mosquitos aegypti A.

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

Recherche

Biologie

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.

Dissection en direct de Drosophile Embryons: méthodes simplifiées pour le dépistage de collections de mutants par immunofluorescence

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.

In vivo d&#39;embryons de drosophile Centrifugation

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.

Livraison bactérienne de l&#39;ARNi Effectors: Transkingdom ARNi

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.

Injection intrapéritonéale en Zebrafish adultes

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.

Imagerie verticale de<em&gt; Drosophila</em&gt; Embryons

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.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

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.

Transfert de gènes dans des embryons de poulet âgés par<em&gt; Ex ovo</em&gt; Électroporation

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.

ARNi Trigger Livraison en<em&gt; Anopheles gambiae</em&gt; pupes

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.