RNAi-vermittelten Gen-Silencing in Mücken, bedarf der vorherigen Herstellung und Reinigung von spezifischen dsRNAs, dass das Gen von Interesse Ziel. Für dsRNA-Synthese, empfehlen wir die Ambion Megascript Kit, Verwendung eines T7-RNA-Polymerase-vermittelte in vitro Transkriptions-Reaktion macht. Für dsRNA Reinigung empfehlen wir die Qiagen RNeasy-Kit. Proben von dsRNA zu quantifizieren und angepasst werden, um 3 ug / ul in Wasser. Andere Materialien, die Sie benötigen sind: spitzen Pinzette, Objektträger aus Glas, Glas Mikrokapillare Nadeln, Mikroinjektor (wir verwenden Drummond Nanoject II), einem Lichtmikroskop vor einem kalten Block, eine überdachte Glas-Petrischale, Papierhandtücher und einen Eimer mit Eis montiert . Sie müssen auch einen Weg, um Mücken und geeignete Behälter für sie zusammen mit einer Nahrungsquelle wie Watte in 10% Saccharose getränkt sammeln. <ol><li> Sammeln Sie Ihre Mücken. Mit einem batteriebetriebenen Aspirator, sammeln die Mücken Sie zu injizieren. Stecken Sie die Düse mit einem Papiertuch oder Baumwolle, so dass keine Moskitos entweichen kann, und entfernen Sie das Reservoir. Da kalte Temperaturen Mücken betäuben, zu begraben das Reservoir in einen Eimer mit Eis und warten 5-10 Minuten für die Moskitos nicht mehr bewegt. Betäubung kann auch, indem das Reservoir in einem kalten Raum oder im Kühlschrank oder über CO 2 erreicht werden. Während dieser 5-10 Minuten, mit dem nächsten Schritt fortfahren. </li><li> Bereiten Sie Ihren Arbeitsbereich. Set eine ebene Fläche kalten Block 2 ° C und Ort ein Glasplättchen auf sie. Dies ist, wo die Mücken Rest, wie Sie sie zu injizieren. Bringen Sie ein Glas Nadel auf die Mikroinjektor und nutzen Sie Ihre Zange an der Spitze der Nadel auf die gewünschte Breite zu brechen. Es ist wichtig, dass die Breite groß genug, um genug Platz für Flüssigkeit durchströmt zu einem vernünftigen Preis ist aber schmal genug, um eine minimale Wunde während der Injektion zuzufügen. Eine gute Nadelspitze wird schmal genug, um leicht verbiegen, aber nicht brechen. Legen Sie Ihre Spritzen an die gewünschte Lautstärke (für RNAi, verwenden wir 69 nL). Tauchen Sie die Spitze des Nadel in Ihrer Probe und ziehen Sie Ihre flüssigen Probe in die Nadel mit den Anweisungen des Herstellers. Die flüssige Probe kann eine dsRNA-Lösung Bakteriensuspension oder andere sein, die Injektion Prozess ist für alle gleich. Embed einer Glas-Petrischale in den Eiskübel so dass der Boden der Schale vollständig Kontaktaufnahme ist das Eis. Hier finden Sie Moskitos setzen, bis Sie bereit sind, sie zu injizieren sind. </li><li> Bereiten Sie Ihre Moskitos. Transfer 50-100 Mücken aus dem Reservoir der Glas-Petrischale. Verwenden Sie Ihre Pinzette, um sie zu einer einzigen Schicht dünner, so dass sicher die meisten haben mit dem kalten Boden der Schale kontaktieren, um sie betäubt. Decken diese Mücken mit der Schale bedecken, wenn nicht in Gebrauch ist. Zurück Reservoir zur Eiskübel zu halten den Rest der Moskitos betäubt. </li><li> Berg Mücken an kalten Block. Verwenden Sie Ihre spitzen Pinzette Moskitos ein zu einer Zeit übertragen aus der Schüssel auf den Objektträger auf dem kalten Block unter dem Lichtmikroskop montiert. Behandeln Sie die Mücken sanft, vorzugsweise an den Beinen. Line up so viele wie 30 Mücken side-by-side auf der Folie, so dass Sie sicher, kann jeder mit der Mikroinjektor bekommen und dafür sorgen, jeder Kontakt mit der kalten Folie. Stellen Sie sicher, Mücken bis sich ordentlich gefüttert, so dass während Sie entlang gehen Sie wissen, welche haben und die nicht injiziert. Lassen Sie andere Mücken in den gekühlten Petrischale abgedeckt. </li><li> Inject Ihre erste Gruppe von Mücken. Halten Sie Ihre spitzen Pinzette kippte in einer Hand und halten Sie die Mikroinjektor in der anderen Hand wie einen Bleistift. Verwenden Sie die Zange, um die Moskitos zu bewegen, wie Sie benötigen (handle ihnen durch die Beine, um Verletzungen zu vermeiden) und zu halten, die Mücke stabil wie Sie spritzen. Dies funktioniert am besten, wenn Sie die Zange zu verwenden, um eine Seite des Moskito-Unterstützung, wie Sie injizieren von der anderen Seite. Schieben Sie einfach die Nadelspitze in Mücke Thorax. Es ist am besten in eine straff noch dünnen Teil der Kutikula, um Verletzungen zu minimieren, zu injizieren. Vermeiden Sie auch Injektion zu tief, Sie wollen nur die Spitze, um im Inneren des Thorax werden; tiefere Injektionen verursachen größere Wunden, die das Überleben gefährden wird. Sobald die Nadelspitze in der Position, drücken und loslassen des Pedals der Mikroinjektor Geräte, die Ihren vorgegebenen Volumen von dsRNA liefern wird. Warten Sie eine Sekunde oder zwei, und entfernen Sie die Nadel aus der Mücke. Wenn ein Tropfen Flüssigkeit wird außerhalb der Wunde, ein paar Sekunden warten, um es in die Wunde aufgenommen werden. Wenn es nicht innerhalb von wenigen Sekunden zu absorbieren ist, zu verwerfen, dass Mücken und versuchen Sie es erneut. Fahren Sie mit der Einspritzung für alle Mücken auf dem Glasträger. </li><li> Speichern Sie Ihre Mücken. Wenn Sie fertig sind, legen Sie Ihre injiziert Mücken in gekennzeichneten Behältern (wir bevorzugen 1 Pint Wachs ausgekleideten Karton Tassen gedeckt durchNetzgewebe und sicherte mit Gummibändern und einem Pappdeckel). </li><li> Weiter Injektionen bis zur fertigen. Continue, um Stechmücken auf der Folie platzieren, injizieren und Transfer zum Bechern, bis Sie die Stichprobengröße Sie Lust haben. Konto für einige Todesfälle-Anfänger haben viele Mücken aus dem Trauma der Injektion sterben. Dies wird mit der Praxis zu verbessern, bis fast 100% der Mücken überleben Injektion. </li><li> Wenn Sie fertig sind, legen Sie ein gefaltetes, nasses Papier Handtuch über den Kelch zusammen mit einer Nahrungsquelle und decken die Tassen mit etwas Feuchtigkeit wie Kunststoff-Folie oder ein Kunststoff-Petri Deckel zu behalten. Injected Mücken sind anfällig für Austrocknung, so dass die Feuchtigkeit, die durch das nasse Handtuch zur Verfügung gestellt und erhalten durch den Kunststoff wird dazu beitragen, dies zu verhindern. </li><li> Zeigen Sie mit der Mücken in einer kontrollierten Umweltbedingungen Kammer für die Inkubation. </li></ol> Hinweis: Effizienz der Gen-Silencing ist sehr variabel und hängt von einer Reihe von Faktoren, einschließlich Transkript und Protein turn-over-Raten und dsRNA Aufnahme Effizienz von Zellen und Organen. Wir haben festgestellt, dass Schweigen so früh wie 1 Tag nach der Injektion beginnt, und kann bis zu 6 Tage nach der Injektion andauern. Verwenden Sie Transkript und Protein Nachweismethoden wie quantitative PCR und Western-Blot zu Schweigen zu validieren. Hinweis: Es gibt mehrere Variationen über mehrere Schritte der Mücke Einspritz-und RNAi-vermittelten Gen-Silencing-Technik. Einige Varianten sind beschrieben durch: Boisson, et al (2006) und Blandin, et al (2002)...

<ol>
	<li>Boisson, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2006).</li><li>Blandin, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2002).</li></ol>

Effizienz der Gen-Silencing ist sehr variabel und hängt von einer Reihe von Faktoren, einschließlich Transkript und Protein turn-over-Raten und dsRNA Aufnahme Effizienz von Zellen und Organen. Wir haben festgestellt, dass Schweigen so früh wie 1 Tag nach der Injektion beginnt und kann bis zu 6 Tage nach der Injektion andauern. Verwenden Sie Transkript und Protein Nachweismethoden wie quantitative PCR und Western-Blot zu Schweigen zu validieren.

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		<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>Megascript</td>
<td>kit</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>for dsRNA synthesis; T7 RNA polymerase-mediated in vitro transcription reaction</td>
</tr>
<tr>
<td>RNeasy</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>For dsRNA purification</td>
</tr>
<tr>
<td>dsRNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>should be quantified and adjusted to 3 &#x03BC;g/ &#x03BC;L in water</td>
</tr>
<tr>
<td>fine-tipped forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass slide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass microcapillary needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microinjector</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>Nanoject II</td>
<td>&nbsp;</td>
<tr>
<td>light microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mounted above a cold block</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>covered, glass</td>
</tr>
<tr>
<td>paper towels</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% sucrose</td>
<td>food</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. gambiae</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
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protocol for rnai assays in adult mosquitoes (a. gambiae)

Reverse-genetische Ansätze haben sich als äußerst nützlich für die Bestimmung, welche Gene zugrunde liegenden Widerstand gegen Vektor Krankheitserreger in Mücken. Dieses Video-Protokoll stellt ein Verfahren durch die Dimopoulos Labor verwendet werden, um dsRNA in Anopheles gambiae Mücken, die den Malaria-Parasiten Hafen zu injizieren. Die Technik die Manipulation der Injektion Setup und Injizieren dsRNA in den Brustkorb dargestellt.

Watch this Scientific Journal Video about Protocol for RNAi Assays in Adult Mosquitoes A. gambiae at JoVE.com

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Protokoll für die RNAi-Assays in Adult Moskitos (A. gambiae)

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

protocol-for-rnai-assays-in-adult-mosquitoes-a-gambiae

Research

JoVE Journal

Biology

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

12.6K Aufrufe.  Johns Hopkins University. Reverse-genetische Ansätze haben sich als äußerst nützlich für die Bestimmung, welche Gene zugrunde liegenden Widerstand gegen Vektor Krankheitserreger in Mücken. Dieses Video-Protokoll stellt ein Verfahren durch die Dimopoulos Labor verwendet werden, um dsRNA in Anopheles gambiae Mücken, die den Malaria-Parasiten Hafen zu injizieren. Die Technik die Manipulation der Injektion Setup und Injizieren dsRNA in den Brustkorb dargestellt.

Serie: Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Protokoll für Dengue-Infektionen in Mücken (Aedes aegypti) und Infektionsbiologie Phänotyp Bestimmung

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Forschung

Biologie

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protokoll für Mosquito Aufzucht (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Protokoll für Plasmodium falciparum-Infektionen in Mücken und Infektionsbiologie Phänotyp Bestimmung

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

Ein besseres Mosquito: Identifizierung der Gene aktivieren Malaria und Dengue-Fieber Widerstand in A. gambiae und A. aegypti Moskitos

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSION esiRNA für RNAi-Screening in Säugerzellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-vermittelte Gen-Knockdown und<em&gt; In Vivo</em&gt; Diurese Assay in Adult Female<em&gt; Aedes aegypti</em&gt; Moskitos

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Ausdauertraining Protokoll und Längs-Performance-Assays für Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Gekoppelt Assays zur Überwachung Proteinrückfaltung in<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Ein Multi-Nachweistest für Malaria tragenden Mücken

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

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 ...