Die Autoren bedanken sich bei Stephen Crews für dysfusion cDNA, Dys-Antikörper und w 1118 fliegt danken.

1. Embryo-Entnahme <ol><li> Set up Käfigen bei 25 ° C mit 2-4 Tage alten w 1118 flies.Grape Saft Platten pro Stunde während des Tages verändert, um die Entnahme der Eizellen über 1-2 Tage vor der Sammlung synchronisieren </li><li> Sammeln Embryonen für 1 Stunde bei 25 ° C </li><li> Schneiden Sie ein rechteckiges Stück von Traubensaft Agar, leicht in der Mitte mit einer Rasierklinge geschnitten, lassen Sie eine Zeile in der Agar- </li><li> Verwenden Sie ein Metall-Sonde Embryonen aus Traubensaft Platte Transfer zu Ihrem Stück Traubensaft Agar, Linie sie in einer geraden Linie mit dem Embryo längere Achse bei 45 Grad-Winkel auf der Linie in der Mitte des Agar. Holen Sie sich eine gerade Linie von etwa 50 Embryonen. Achten Sie darauf, alle Embryonen in die gleiche Richtung, mit hinteren Ende zeigt weg von der Linie. </li><li> Schneiden Sie ein rechteckiges Stück Doppel-Klebeband, und legen Sie es zu einem 18x18mm Deckglas. </li><li> Pick up Embryonen mit Doppel-Klebeband auf das Deckglas </li><li> Legen Sie das Deckglas auf einen Objektträger, trocken der Embryo in der Luft für ca. 10 min. </li><li> Bedecken Sie den Embryo mit einer dünnen Schicht von 700 Halocarbonöl, jetzt ist es fertig zur Injektion. </li></ol> 2. Pulling Needle Machen Mikroinjektion Nadeln, indem Glaskapillaren. Wir verwenden eine Nadel puller von World Precision Instruments (Modell PUL-1). Die Nadel-Abzieher-Programm Spezifikationen sind: Wärme: 1, Delay 4. 3. Füllen des Needles Back-Belastung Nadeln mit der Eppendorf-Pipette mit p20 Eppendorf Microloader Tipps ausgestattet, in der Regel laden 1,5-2 ul dsRNA 4. Break the Needles <ol><li> Bevor Sie das Nadel, statt einem Deckglas auf einem Objektträger. </li><li> Bereiten Sie eine Folie mit einem Tropfen Öl in der Mitte des Schlittens </li><li> Schalten Sie den Picospritzer III picopump, um den Luftdruck einstellen </li><li> Break the gefüllt Nadel gegen die Kante eines Deckglases, wodurch eine scharfe Spitze. </li><li> Wenn die Spitze gebrochen ist, Schritt auf dem Fußpedal von Picospritzer III picopump, werden einige dsRNA austreten der Nadelspitze. </li><li> Nehmen Sie die Folie mit dem Deckglas. </li><li> Legen Sie die Nadel unter die Öltropfen in der Mitte der Folie vorbereitet, bevor. </li><li> Passen Sie die Einstellung auf dem Picospritzer III picopump bis 100-200PL dsRNA erhalten, wenn Sie auf das Fußpedal Schritt. dsRNA kann als kleine Tröpfchen zu sehen. </li></ol> 5. Injektion <ol><li> Bringen Sie die Nadelspitze auf den hinteren Teil des ersten Blastoderm Embryo in der gleichen Bildebene und spritzen den Embryo aus der Mitte rund 30-50% Ei Länge. </li><li> Nachdem Sie auf das Fußpedal der Picospritzer III picopump Schritt wird eine kleine Menge an dsRNA als kleiner Punkt in der Injektionsstelle auftreten. </li></ol> 6. Phänotypische Analyse <ol><li> Nach der Injektion, legen Sie die Folie in einem überdachten feuchten Kammer (Kunststoff-Petrischale) bei 18 ° C, bis die Embryonen, um die gewünschte Embryonalstadium entwickeln. </li><li> Verwenden Sonde Embryonen aus dem doppelseitigen Klebeband entfernen und schieben Sie sie an den Rand des Deckglases. Waschen Sie die Embryonen aus der Folie in eine Sammlung Fläschchen mit Nylon-Mesh an der Unterseite mit Heptan, 3 x waschen mit PBT. Dechorionate Embryonen in 50% Bleichmittel für 5 min und 3 x waschen mit PBT. Transfer-Embryonen aus der Sammlung Fläschchen, ein Stück Nylon-Mesh, und dann tauchen die Nylon-Mesh in die Fixierlösung (5ml Heptan, 4,5 ml PBS, 0,5 ml 37% Formaldehyd) zu Embryonen freizugeben. Fix Embryonen in die Fixierlösung für 30min. Devitellinize Embryonen mit Methanol-Embryonen zu einem Eppendorf-Röhrchen, und immunostain die Embryonen unter Verwendung von Standard-Protokollen. </li><li> Embryonen können auch geeignete Larvenstadium entwickeln. </li><li> Bereiten Sie eine Folie mit einem Rückgang von Halocarbon 700 Öl in der Mitte </li><li> Transfer ein Larven auf die Folie, legen ein Deckglas auf dem Gipfel </li><li> Beachten Larven unter Hellfeld-Mikroskop. </li></ol> 7. Repräsentative Ergebnisse: Ein Beispiel für dsRNA knock down dysfusion (dys-) Gen in Drosophlia Embryo ist in Fig1 gezeigt. GFP-dsRNA injizierten Embryonen sind die Negativ-Kontrolle. Dys ist ein bHLH PAS Transkriptionsfaktor in trachealen Fusion exprimiert. GFP-dsRNA injizierten Embryonen zeigen normale Luftröhre Fusion und Dys Protein in Fusion-Zellen (Abbildung 1A). Auf der anderen Seite, injiziert dys-dsRNA Embryonen zeigen gescheitert Filiale Fusion und Dys-Proteine ​​sind nicht in Fusion-Zellen (Abbildung 1B). Wenn dsRNA injizierten Embryonen zu Larvenstadium entwickeln, zeigten die dys-dsRNA injiziert Larven gescheitert Filiale Fusion (Fig.1D) im Vergleich zu GFP-dsRNA injiziert negative Kontrolle (Abbildung 1C). Diese Ergebnisse zeigten, effektive knock down der endogenen dys Genexpression durch dsRNA-Injektion in Drosophila-Embryonen <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Beseitigung von Dys-Funktion durch dys-RNAi zeigt tracheale Fusion defects. Blastoderm Embryonen (w 1118) wurden entweder mit GFP-dsRNA (negative Kontrolle) oder dys-dsRNA injiziert und untersucht für Luftröhrendefekten. (A) 16. Etappe Embryo mit GFP-dsRNA injiziert und gefärbt mit α-Dys-und MAb 2A12, die Flecken der Tracheallumen. Prominent angezeigt wird die seitliche Rumpf, der hat fusioniert. Pfeile deuten auf seitliche Rumpf Dys-positive Zellen Fusion. (B) 16. Etappe Embryo mit dys-dsRNA injiziert und gefärbt mit α-Dys-und MAb 2A12. Der Embryo zeigte einen Mangel an seitlichen Kofferraum Fusion (die Sternchen zeigt die normale Lage eines lateral-Stamm Fusion unter Kontrolle Embryonen). Keine Dys-positive Zellen beobachtet, was darauf hinweist, dass die dys-RNA effektiv abgeschafft Dys-Protein. (C bis D) Second-Larvenstadium entweder mit GFP-dsRNA oder dys-dsRNA wurden von Hellfeld-Mikroskopie für Luftröhrendefekten untersucht. (C) Websites der Fusion sind mit einem Sternchen in GFP-dsRNA injiziert Kontrolle Larven angegeben. (D) Die seitliche Rumpf in der dys-RNA injiziert Larven Sicherung fehlgeschlagen ist, zeigt (*) die normale Lage eines seitlichen Kofferraum Fusion.

<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>

Die dsRNA Injektionsverfahren hier anwesend ermöglicht eine sehr sensible und schnelle Analyse der Genfunktion in Drosophila trachealen Entwicklung. Diese Methode kann möglicherweise angewendet Genfunktion für andere Gewebe-und Organentwicklung zu analysieren.

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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
<td>&nbsp;</td>
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<td>Picospritzer III picopump</td>
<td>&nbsp;</td>
<td>Parker Precision Fluidics</td>
<td>051-0500-900</td>
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rnai interference by dsrna injection into drosophila embryos

Genetisches Screening ist eine der leistungsstärksten Methoden zur Einblicke in komplexe biologische Prozess 1. Im Laufe der Jahre viele Verbesserungen und Werkzeuge zur genetischen Manipulation geworden in Drosophila 2 zur Verfügung. Bald nach der ersten Entdeckung von Frie-und Mello 3, dass doppelsträngige RNA verwendet werden, um knockdown die Aktivität einzelner Gene in Caenorhabditis elegans, der RNA-Interferenz (RNAi) gezeigt, dass eine leistungsstarke Reverse genetische Ansatz bieten, um Genfunktionen in Drosophila Orgel Entwicklung zu analysieren war sein 4, 5. Viele Organe, einschließlich Lunge, Niere, Leber und Kreislauf-System sind von verzweigten röhrenförmigen Netzwerken zusammen, dass der Transport lebenswichtigen Flüssigkeiten oder Gase 6, 7. Die Analyse von Drosophila tracheale Bildung bietet ein exzellentes Modellsystem zur Morphogenese von anderen röhrenförmigen Organen 8-Studie. Die Berkeley Drosophila-Genom-Projekt hat hunderte von Genen, die in den Tracheen exprimiert werden aufgedeckt. Zur Untersuchung der molekularen und zellulären Mechanismen der tube formation, ist die Herausforderung, die Rolle dieser Gene in trachealen Entwicklung zu verstehen. Hier haben wir beschrieben, eine detaillierte Methode der dsRNA-Injektion in Drosophila-Embryo den Zuschlag einzelner Gen-Expression. Wir haben erfolgreich niedergeschlagen endogenen dysfusion (Dys) Genexpression durch dsRNA Injektion. Dys ist ein bHLH-PAS-Protein in trachealen Fusion exprimiert, und es ist für die tracheale Filiale Fusion 9, 10 erforderlich. dys-RNAi vollständig eliminiert dys Ausdruck und führte in trachealen Fusion Defekt. Diese relativ einfache Methode stellt ein Werkzeug, um Gene für tissure und Organentwicklung in Drosophila requried identifizieren.

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

RNA-Interferenz wurde als sehr effektiv erwiesen, um Genfunktionen in Analyse<em&gt; Drosophila</em&gt; Trachealen Entwicklung. Ein detailliertes Protokoll von Jiang Labor verwendet werden, um dsRNA zu fliegen Embryonen knockdown Genexpression injizieren dargestellt. Diese Technik hat das Potenzial für das Screening von Genen für die Gewebe-und Organentwicklung in erforderlich<em&gt; Drosophila</em&gt;.

RNAi-Interferenz durch dsRNA Injektion in 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 ...

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

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K Aufrufe.  Oakland University. RNA-Interferenz wurde als sehr effektiv erwiesen, um Genfunktionen in Analyse Drosophila Trachealen Entwicklung. Ein detailliertes Protokoll von Jiang Labor verwendet werden, um dsRNA zu fliegen Embryonen knockdown Genexpression injizieren dargestellt. Diese Technik hat das Potenzial für das Screening von Genen für die Gewebe-und Organentwicklung in erforderlich Drosophila.

Serie: 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.

Die Injektion von dsRNA in Female A. aegypti Mosquitos

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

Forschung

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.

Live-Präparation Drosophila Embryos: Optimierte Verfahren für das Screening Mutant Sammlungen von Antikörper-Färbung

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-Zentrifugation von Drosophila Embryonen

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.

Bakterielle Lieferung von RNAi Effektoren: 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.

Intraperitoneale Injektion in Adult Zebrafisch

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.

Upright Imaging von Drosophila Embryos

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.

Primären Zellkulturen aus Drosophila Gastrula Embryonen

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.

Gentransfer in Hühnerembryonen durch Ältere<em&gt; Ex ovo</em&gt; Die Elektroporation

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-Trigger Lieferung in<em&gt; Anopheles gambiae</em&gt; Pupae

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.