A. propagar el virus en la línea celular C6/36. <ol><li> Las células crecen un 80% de confluencia en 75 cm 2 frasco; </li><li> Retire el papel con 3.4 ml restantes en el frasco; </li><li> Tomar 0,5 ml de virus de archivo y añadir en la celda de arriba con la multiplicidad de infección de 1,5 partículas virales por célula. Agitando el matraz durante 15 minutos lentamente a la temperatura ambiente. </li><li> Incubar durante 45 minutos a 5% de CO 2 y 37 ° C </li><li> Añadir 30 ml de medio, y se incuba durante 5 días. </li></ol> B. Prepare la mezcla de virus y la sangre <ol><li> Separe la celda con el raspador y la transferencia de la célula y los medios de comunicación a los 50 tubos cónicos mL; </li><li> Centrifugado a 800g durante 10 minutos, recoger el sobrenadante, pero deje 1 ml de sobrenadante con el pellet de células; </li><li> Congelar el sedimento celular por encima de las emisiones de CO 2 en seco y luego descongelar a 37 ° C baño de agua, repetir dos o tres veces; </li><li> Centrifugado a 800g durante 10 minutos, tome el sobrenadante, y se combinan con el sobrenadante se recogió desde el paso 3; </li><li> Recoger la sangre humana entera con el mismo procedimiento (paso 3) para la preparación de la cultura de gametocitos de infectar a los mosquitos Anopheles con Plasmodium falciparum; </li><li> Combine la misma cantidad de lo anterior la sangre humana entera con sobrenadante de virus desde el paso 4, y añadir suero humano (10% del volumen total). </li><li> Incubar la mezcla por encima de la sangre humana y el virus del dengue durante 30 minutos a 37 ° C baño de agua </li></ol> C. mosquitos Alimentación Este procedimiento es casi idéntico a lo que se han descrito en la sección de infecciones por Plasmodium falciparum en los mosquitos. Nos ensayo de la infección del intestino medio en el día 7, y la infección por la saliva a los 14 días después de que se alimentan de sangre. Todo el material que entró en contacto con el virus se trata en primer lugar con el 75% de etanol y luego con lejía al 10%. D. Ensayo el título del virus en los tejidos del mosquito <ol><li> Dos o tres días antes de la disección de los tejidos del mosquito, crecer C6/36 células en una placa de 24, así como que las células alcanzan el 80% de confluencia en el día en que el ensayo se lleva a cabo; </li><li> Los mosquitos son anestesiados a 4 ° C, sacrificado y superficie estéril por inmersión en etanol al 75% durante 1 minuto. A continuación, los mosquitos se lavaron con agua estéril dos veces antes de la disección de las glándulas salivales del intestino medio o bajo el microscopio de disección. A partir de este paso, todo el procedimiento que figura a continuación se llevó a cabo en un ambiente estéril, como una cabina de seguridad biológica. Estos tejidos se transfiere a un tubo que contiene 150 l de medios y homogeneizado con una pastilla Kontes mortero motor durante 90 segundos; </li><li> Realizar una dilución al 1:100 añadiendo 10 l de la homogeneizado en 990 medios de comunicación l; </li><li> Hacer más de 1: 10, 1:100 y 1:1000 dilución con el medio de la placa de 96 pozos; </li><li> Retire el papel de la placa de 24 pocillos, y añadir 100 ml de cada una de las cuatro disoluciones correspondientes a cada pocillo; </li><li> Agitar la placa lentamente durante 15 minutos a la temperatura ambiente, luego se incuban durante 45 minutos a 5% de CO 2 y 37 ° C; </li><li> Añadir 1 ml de Metilcelulosa superposición (1%) a cada pocillo; </li><li> Incubar las placas a 37 º C con CO2 al 5% durante 5 días; </li><li> Los pasos de aquí en adelante no requieren de un ambiente estéril, pero como con cualquier otro agente patógeno de cuidado se debe tomar en todo momento. Deseche superposición Metilcelulosa de cada placa y borrar para eliminar el exceso de material </li><li> Añadir 1 ml de solución fijadora metanol / acetona (1:1) a cada pocillo. Mantener a 4 ° C durante 1 hora; </li><li> Vierta el fijador, y lavar una vez con PBS 1X; </li><li> Hacer la dilución 1:1000 a la primaria de anticuerpos contra el virus con un 5% Blotto; </li><li> Añadir 200 l de fluido hiperinmune diluido a cada pocillo, incubar a 37 ° C durante 1 hora; </li><li> Extraer el líquido hiperinmune y la placa de lavar con PBS 1 X </li><li> Prepare una dilución 1:1000 de cabra anti-ratón conjugado (Cat. KPL # 074 a 1806) en BLOTTO del 5% (leche descremada en polvo 5 g en 100 ml de 1 X PBS), a continuación, añadir 200 l en cada pocillo e incubar a 37 ° C durante 1 hora; </li><li> Prepare el sustrato de la siguiente manera: añadir 1 tableta de 3&#39;-Diaminobencidina terrahydrochloride (Cat. Sigma # D5905) en 20 ml de 1 X PBS, después de disolverse, y se añaden 8 l de peroxidasa de hidrógeno al 30% </li><li> Añadir 200 l de sustrato por encima a cada pocillo. Deje reposar a temperatura ambiente durante 10 minutos </li><li> Retire del sustrato y detener la reacción mediante la adición de 1 ml de agua destilada a cada pocillo </li><li> Vierta en platos de agua y secarlos a secas </li><li> Contar con la partícula del virus </li></ol>

<!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>Incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with 5% CO2, 37oC</td>
</tr>
<tr>
<td>50 mL conical tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>plastic</td>
</tr>
<tr>
<td>human serum and blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+</td>
</tr>
<tr>
<td>pipette tip</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture </td>
</tr>
<tr>
<td>pipettman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipetts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass, sterile</td>
</tr>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>37C</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>circulating water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass membrane feeders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with rubber tubing to fit feeders</td>
</tr>
<tr>
<td>mosquito cups</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75% ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-culture plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>6-well, 24-well and 96-well plates</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>fine-tip forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X, sterile</td>
</tr>
<tr>
<td>dissecting microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>C6/36 cells</td>
<td>cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For virus propagation</td>
</tr>
<tr>
<td>C6/36 medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>MEM with 10% heat inactivated FBS, 1% L-glutamine and non-essential amino acid and 1% (v/v) Penicillin-Streptomycin.</td>
</tr>
<tr>
<td>3&#x2019;-Diaminobenzidine terrahydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>1 tablet in 20 ml of 1 X PBS, dissolve and then add 8 &#181;l of 30% hydrogen peroxidase</td>
</tr>
<tr>
<td>30% hydrogen peroxidase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. aegypti</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito</td>
</tr>		</tbody>
		</table>
		</div>

protocol for dengue infections in mosquitoes (a. aegypti) and infection phenotype determination

El propósito de este procedimiento es para infectar a los mosquitos Aedes con el virus del dengue en una condición de laboratorio y examinar el nivel de infección y dinámica de los virus en los tejidos del mosquito. Este protocolo se utiliza rutinariamente para el estudio de las interacciones entre el virus de mosquitos, especialmente para la identificación de los factores del huésped novela que son capaces de determinar la competencia del vector. El experimento debe ser realizado en un laboratorio BSL2. Al igual que en las infecciones por Plasmodium falciparum, vestimenta adecuada, guantes y bata de laboratorio se debe usar en todo momento. Después del experimento, todos los materiales que entraron en contacto con el virus deben ser tratadas con etanol 75% y se blanquea antes de proceder con el lavado normal. Todos los otros materiales deben ser esterilizados en autoclave antes de desecharlos.

Watch this Scientific Journal Video about Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination at JoVE.com

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

Una vez que un gen es identificado como potencialmente resistente a los virus del dengue, que debe ser evaluado por su papel en la prevención de las infecciones virales en el mosquito. Este protocolo se ilustra cómo la extensión de infecciones de dengue de los mosquitos pueden ser analizados. Las técnicas para el cultivo de los virus en la cultura, la alimentación de los mosquitos membrana de sangre humana, y analizando los títulos virales en el intestino del mosquito se ha demostrado.

Protocolo para las infecciones de dengue en mosquitos (Aedes aegypti) y determinación de fenotipo infección

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 ...

protocol-for-dengue-infections-mosquitoes-aegypti-infection-phenotype

Research

JoVE Journal

Biology

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

17.4K vistas.  Johns Hopkins University. Una vez que un gen es identificado como potencialmente resistente a los virus del dengue, que debe ser evaluado por su papel en la prevención de las infecciones virales en el mosquito. Este protocolo se ilustra cómo la extensión de infecciones de dengue de los mosquitos pueden ser analizados. Las técnicas para el cultivo de los virus en la cultura, la alimentación de los mosquitos membrana de sangre humana, y analizando los títulos virales en el intestino del mosqui...

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

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Microinyección de A. aegypti embriones para obtener los mosquitos transgénicos

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Investigación

Biología

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.

Protocolo de infecciones por Plasmodium falciparum en la determinación de fenotipo mosquitos y la infección

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

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

La disección de las glándulas salivales y del intestino medio de Ae. aegypti

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

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

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 Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

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 Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocolo para ensayos de RNAi en mosquitos adultos (A. gambiae)

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

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

Cómo prevenir la propagación de la malaria y el dengue Con mosquitos genéticamente modificados

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

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.

La construcción de un mosquito mejor: La identificación de los genes que permiten resistencia a la malaria y el dengue en A. gambiae y A. aegypti

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

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 mediada por Gene y Derribo<em&gt; En Vivo</em&gt; Ensayo de diuresis en Mujeres Adultas<em&gt; Aedes aegypti</em&gt; Los mosquitos

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

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un Protocolo de vídeo de la infección retroviral en Primaria Intestinal organoide Cultura

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Mantener los mosquitos Aedes aegypti infectados con Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...