Este trabajo fue apoyado por subvenciones del NIAID (R01AI074951, U54AI057168) y el Penn Genoma Fronteras Instituto de Carolina del Sur, de los NIH T32HG000046 de TSM, de los NIH y T32GM07229 T32AI007324 a LRS.

Parte 1: RNAi en 384 placas <ol><li> La estandarización de los reactivos utilizados para la investigación es esencial. Probamos lotes individuales de los medios de comunicación de Schneider, suero fetal bovino, y reactivos de coloración. Luego alícuota y el uso del mismo lote de toda la pantalla. </li><li> Para cualquier ensayo, lo mejor es probar varias líneas celulares diferentes para determinar cuál es más susceptible a la biológica de lectura. Por lo general el uso de la línea S2-DRSC. </li><li> Se cultivan las células Drosophila a 25 ° C en los medios de comunicación completa de Schneider. <ol class="lalpha"><li> Schneider medios de comunicación </li><li> 10% de SFB inactivado por calor </li><li> 1X L-glutamina </li><li> 1X penicilina / estreptomicina los antibióticos </li></ol></li><li> Las células de Drosophila son semi-adherente y se pasan sin tripsinización cada cuatro días con la pipeta las células de los frascos y la dilución de las células de 1:10 en un matraz fresco. Las células deben estar en fase de registro para los experimentos. </li><li> Placa a placa y la variabilidad del día a día se reducen al mínimo mediante el uso de manejo de líquidos automatizados para todas las adiciones a varios líquidos y placas. Utilizamos un WellMate (Matrix). </li><li> Preparar tubos WellMate. <ol class="lalpha"><li> Spray campana y WellMate con etanol al 70%. </li><li> Remueva el tubo de empaque y rociado con etanol al 70%. </li><li> Inserte un cassette tubería en la dispensación de la posición en la WellMate. </li><li> El primer tubo con 25 ml de etanol al 70%. </li><li> Esterilice los tubos en etanol durante 15 minutos, el entonces primer con otro de 25 ml de etanol. </li><li> Enjuague la tubería de la preparación con 50 ml de PBS estéril. </li></ol></li><li> Separar las células del frasco y el recuento. Precipitar las células para que tenga un 25% más de lo necesario (300xg, 5 min). El número de células sembradas por pocillo debe ser optimizada de acuerdo a la longitud de ensayo. Al llevar a cabo análisis automatizado de imágenes, una sola monocapa sin grumos de células es ideal para la segmentación de la imagen. </li><li> Volver a suspender las células sedimentadas en el suero libre de Schneider en los medios de comunicación 1.7x10 6 células / ml. </li><li> Realizamos la pantalla en 384 placas pre-alícuotas con una biblioteca disponible en el mercado de dsRNA a una concentración de 250ng/well. Varios pozos de cada placa se deja en blanco para los controles, que se añaden manualmente. Usamos dsRNA a luciferasa como control negativo. Usamos dsRNA contra Thread (DIAP) como control de sólidos RNAi. Derribar los resultados de este factor anti-apoptóticos de la muerte celular dramático y es una manera rápida visual para evaluar la calidad de la derribarlo. Por último, se utiliza un dsRNA viral contra el periodista, en este caso de beta-galacosidase, como un control positivo para validar que pueden inhibir la infección viral con nuestro ensayo de lectura. Si el goteo dsRNA en el plato en el momento de la siembra de células, una alícuota de dsRNA l en la esquina del pozo para permitir una mejor visualización, centrifugar dsRNA contra el fondo del pozo (300xg, 1 min) antes de añadir las células. </li><li> Use WellMate añadir 10 celdas l preparada anteriormente a cada pocillo. </li><li> Girar las células en las placas mediante la centrifugación de 300xg, 1 min. </li><li> Incubar en 25 ° C incubadora durante 45 minutos. </li><li> Añadir 20 l completo de Schneider medios de comunicación y centrifugar 300xg, 1 min. </li><li> Coloque las placas en humidificado recipientes Tupperware cubierto con toallas de papel empapado en agua. El volumen de un 384 y es pequeña, por lo tanto, la evaporación puede tener grandes efectos en la biología, que a su vez puede conducir a cambios de artefactos en las salidas de lectura de los pozos de borde. Para superar esto, las placas deben mantenerse en condiciones de humedad alta. </li><li> Incubar durante tres días para permitir la caída fuerte. No hay necesidad de cambiar los medios de comunicación o de abrir los contenedores humidificado durante este tiempo. </li></ol> Parte 2: Infección con el virus vaccinia <ol><li> Esterilizar multi-canal de aspiración en Quatricide durante 15 minutos, luego enjuague con etanol al 70%. </li><li> Prepare WellMate como se describió anteriormente. </li><li> Prepare los medios de comunicación de Schneider con FBS al 2% para la infección mediante la dilución de 1:05 completar medios de comunicación en medio libre de suero. </li><li> Acciones filtro de virus crudo a través de una jeringa 0,8 um filtro para eliminar los desechos celulares o el uso de virus purificado. </li><li> Diluir el virus vaccinia en el suero de Schneider 2% los medios de comunicación para obtener la multiplicidad de infección (MOI), de 2. </li><li> Quitar de la tubería WellMate PBS, e imprima con virus diluido hasta que el tubo esté libre de burbujas de aire. </li><li> Use esterilizados multi-canal de aspiración y colector de vacío para extraer con cuidado los medios de comunicación de las placas de dsRNA tratados con células de Drosophila. </li><li> Prescindir de 30 virus por pocillo en placas con el WellMate. </li><li> Centrifugar las placas 1 minuto a 300xg. </li><li> Esterilizar tubos WellMate. Primer con 25 ml de 10% bleach, espere 10 minutos, y la primera con otro de 25 ml de lejía al 10%. </li><li> El primer tubo con 50 ml de agua, seguido de 50 ml de etanol al 70%. Volver a la tubería de su embalaje. </li><li> Volver a las placas humidificado recipiente Tupperware. Se incuba a 25 ° C durante 48 horas. </li></ol> Parte 3: Placas de tinción <ol><li> Fijar placas en 15 l de formaldehído al 4% / PBS durante 10 minutos. Líquido se elimina con un aspirador de multi-canal y colector de vacío a cada paso. Una vez que las placas se han fijado, equipos y reactivos estériles ya no son necesarios. </li><li> Quitar reparar y utilizar WellMate añadir 30 l PBST (PBS + 0,1% Triton-X). Incubar 10 minutos y repita. Operar WellMate como se describió anteriormente, excepto el tubo no necesita ser esterilizado. </li><li> Para detectar la infección por vaccinia se utiliza un reportero β-galactosidasa impulsado por un promotor temprano / tardío vaccinia. </li><li> Eliminar las células de líquido y se incuba en el bloque (PBST con un 2% BSA) durante 10 minutos. </li><li> Diluir el anticuerpo primario en el bloque, eliminar el líquido y añadir 15 l de anticuerpos a cada pipeta repetir así el uso de múltiples canales (Matrix). </li><li> Girar las placas (300xg, 1 min), cubierta con una etiqueta clara, e incubar a 4 ° C durante la noche. </li><li> Eliminar el anticuerpo primario, y el uso de la WellMate para lavar los pocillos 3 veces con PBST, a 10 minutos cada uno. </li><li> Diluir el anticuerpo secundario marcado con fluorescencia (FITC) y la tinción nuclear en el bloque, y añadir 15 l de pozos utilizando una pipeta multicanal repetir. </li><li> Incubar las placas 1 hora a temperatura ambiente, protegido de la luz. </li><li> Lave los pozos en PBST 3 veces, 10 minutos cada uno con el WellMate. </li><li> Selle la placa con la etiqueta engomada transparente, tapar y guardar a 4 ° C hasta tres semanas. </li></ol> Parte 4: Imágenes y Análisis de Imágenes <ol><li> Utilizar un microscopio automatizado a 20X a las placas de la imagen. </li><li> Capturar imágenes de los núcleos total (DAPI) y las células infectadas (FITC). Optimizar el tiempo de exposición para cada canal por separado con el fin de maximizar el rango dinámico. </li><li> Ajustar la función de enfoque automático mediante la adopción de una pila Z de imágenes, 1um separados, que abarcan los aviones por encima y por debajo del plano de espera de atención. Una vez que el plano focal óptima se encuentra, ajustar la configuración de enfoque automático en consecuencia. </li><li> La imagen de toda la placa, la captura de al menos tres imágenes por bien tanto en el canal de Hoechst (DAPI) y el canal de virus (FITC). Elegir los sitios de distintas regiones dentro de un pozo con el fin de capturar las imágenes que representan el bien en su conjunto. </li><li> Use MetaXpress software para escribir un diario para segmentar la imagen y el uso de módulos de Conde núcleos para calcular el número total de células positivas para la tinción nuclear (el total de células) y la infección (FITC) para cada plato. </li><li> Calcular la infección por ciento (100 * Las células FITC positivos / total) y el registro de transformar. </li><li> Para identificar a los candidatos que utilizan un sólido puntaje Z basada en la mediana y rango intercuartil de cada plato. Este método no es sensible a los valores atípicos y los datos no simétrica, y por lo tanto proporciona una mayor potencia en la identificación de éxitos, incluso débil o moderada 10. Para cada plato, la mediana se resta de los datos de transformación logarítmica. A continuación, los valores resultantes se dividen por 0,74 veces el rango intercuartil (la diferencia entre los valores de la percentil 75 y el percentil 25). El factor 0,74 se utiliza para aproximar una distribución normal estándar. </li><li> El robusto puntaje Z es una medida de la distancia en desviaciones estándar de la mediana de la placa. Por ejemplo, un robusto Z-score de 2 indica que la infección% del pozo es de ~ 2 desviaciones estándar de la mediana o la placa de p &lt;0,05. </li><li> Analizar el número de células para identificar a los candidatos citotóxicos. Calcular el puntaje Z de robusta cuenta nucleares. Los candidatos con una sólida Z &lt;-2 en pantallas duplicadas son consideradas citotóxicos. </li><li> Los pozos que tienen un sólido puntaje Z para la infección por ciento de&gt; 2 o &lt;-2 en pantallas duplicadas (p &lt;0,0025), sin citotoxicidad se consideran candidatos potenciales de la pantalla principal. </li><li> Candidatos positivos son validados utilizando reactivos independientes. dsRNAs se generan en contra de los genes de interés de otra región del ARNm y probado que el anterior. </li><li> Los genes de los cuales dos dsRNAs independientes tienen el mismo fenotipo puede ser considerado para el estudio adicional. </li></ol> Parte 5: Imágenes Representante e interpretación de las tasas de infección <img src="/files/ftp_upload/2137/2137fig1.jpg" alt="Figura 1" /> Los pozos de la figura 1. Infectadas no muestran manchas de proteínas de virus de la vacuna, mientras que un representante de infectados y contiene células con tinción positiva para la vacuna-expresó beta-galactosidasa (βgal) de proteínas, medida por microscopía de inmunofluorescencia.Virus = verde, azul = núcleos. <img src="/files/ftp_upload/2137/2137fig2.jpg" alt="Figura 2" /> Figura 2. Software automatizado de análisis de imágenes cuantifica la infección en cada imagen con los parámetros establecidos por el usuario. En una imagen representativa, el número total de núcleos celulares y el número de células que expresan el antígeno viral se contabilizan en función del tamaño y la intensidad de la tinción por encima de la tinción de fondo local. <img src="/files/ftp_upload/2137/2137fig3.jpg" alt="Figura 3" /> Figura 3. Derribo de hilo (DIAP) sirve como un control positivo para el agotamiento de RNAi robusta de los genes diana. Caída de este factor anti-apoptóticos conduce a la muerte celular dramático. Núcleos = azul. <img src="/files/ftp_upload/2137/2137fig4.jpg" alt="Figura 4" /> Derribo Figura 4. De la luciferasa sirve como control negativo para el efecto de la doble cadena de ARN en el tratamiento de la infección. El agotamiento de la luciferasa no tiene ningún efecto sobre la infección en relación con las células no se tratan. Virus = verde, azul = núcleos. <img src="/files/ftp_upload/2137/2137fig5.jpg" alt="Figura 5" /> Figura 5. Derribo de la proteína βgal sirve como un control positivo de una reducción de la infección, ya que el ensayo utiliza los niveles de proteína βgal como una lectura de la infección. El agotamiento de los resultados βgal en una disminución en el porcentaje de células infectadas. Virus = verde, azul = núcleos. <img src="/files/ftp_upload/2137/2137fig6.jpg" alt="Figura 6" /> Figura 6. Derribo de celulares factor de Rab5 resulta en una disminución en el porcentaje de células infectadas. Desde Rab5 se sabe que participan en la endocitosis, este factor probablemente contribuye a la entrada del virus vaccinia. Esto representa un valor atípico ejemplo de la pantalla. Virus = verde, azul = núcleos.

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

Todo el genoma de Drosophila RNAi de detección proporciona un método robusto y eficiente para el examen de los componentes celulares de la infección viral. Un número de virus que infectan a las células de mamíferos Drosophila, que puede ser utilizado para identificar los componentes de la respuesta inmune del huésped intrínseco, y los factores del huésped proteínas necesarias para la replicación viral que puede representar nuevas dianas terapéuticas. Antes de realizar una pantalla, el ensayo debe ser cuidadosamente optimizado para el tipo de células, el número de células, y el nivel de infección, y debe estar bien controlado, tanto positivos como negativos virales y dianas celulares 11. Es importante garantizar una separación suficiente entre los controles positivos y negativos previos a la selección para maximizar el rango dinámico de la prueba. Una vez que la pantalla se ha realizado, los candidatos se pueden dividir en categorías funcionales para determinar qué mecanismos de acogida contribuyen a la infección. De virus de mamíferos, tales como la vacuna, la contribución de los homólogos mamíferos de RNAi se debe determinar como parte del análisis secundario. En conjunto, estos métodos de detección robusta nos permitirá comprender mejor los complejos mecanismos por los cuales los virus interactúan con las células de su huésped.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
<td>&nbsp;</td>
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<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin (pen/strep)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178</td>
<td>&nbsp;</td>
<tr>
<td>Screening</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Genome-wide dsRNA library</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>384 well plates, black with clear bottom</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3712</td>
<td>&nbsp;</td>
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<td>Aluminum seals</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>6569</td>
<td>&nbsp;</td>
<tr>
<td>Clear seals</td>
<td>&nbsp;</td>
<td>Denville</td>
<td>B1212-4</td>
<td>&nbsp;</td>
<tr>
<td>Well Mate</td>
<td>&nbsp;</td>
<td>Matrix</td>
<td>201-10001</td>
<td>&nbsp;</td>
<tr>
<td>24 pin manifold</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>3-000-101</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B2261</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescently-labeled secondary antibodies</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ImageXpressMicro automated microscope</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MetaXpress image analysis software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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rnai screening for host factors involved in vaccinia virus infection using drosophila cells

Patógenos virales representan una amenaza para la salud pública, no sólo puede causar epidemias de virus natural de la enfermedad humana, pero su uso potencial en bioterrorismo es también una preocupación. Una mejor comprensión de los factores celulares que la infección por el impacto que faciliten el desarrollo de la muy necesaria la terapéutica. Los avances recientes en el ARN de interferencia (RNAi), junto con la tecnología de secuenciación completa del genoma de varios organismos ha llevado a la optimización de las pantallas de todo el genoma, a base de células de pérdida de función. Células de Drosophila son particularmente susceptibles a las pantallas a escala del genoma debido a la facilidad y eficacia de RNAi en este sistema 1. Es importante destacar que una amplia variedad de virus que pueden infectar a las células Drosophila, incluyendo un número de virus de mamíferos de importancia médica y agrícola 2,3,4. Anterior pantallas de RNAi en Drosophila se han identificado los factores del huésped que se requieren para diversas etapas de la infección por virus, incluida la entrada, la traducción y la replicación del ARN 5. Por otra parte, muchos de los factores celulares necesarios para la replicación viral en cultivo de células de Drosophila también son limitantes en las células humanas infectadas con estos virus 4,6,7,8, 9. Por lo tanto, la identificación de los factores del huésped cooptado durante la infección viral se presentan nuevos objetivos para la terapia antiviral. Aquí se presenta un protocolo generalizado de una pantalla de alto rendimiento RNAi para identificar los factores celulares implicados en la infección viral, utilizando virus vaccinia como un ejemplo.

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Nuevos factores de acogida implicados en la infección viral puede ser identificado a través de celulares basados ​​en el genoma pérdida de la función de detección de RNAi. A<em&gt; Drosophila</emCelular&gt; modelo de cultivo es particularmente susceptible a este enfoque debido a la facilidad y la eficiencia de RNAi. Aquí se demuestra que esta técnica con<em&gt; Vaccinia</em&gt; Virus como un ejemplo.

RNAi detección de los factores del huésped implicados en Vaccinia Infección del virus utilizando Drosophila Celdas

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

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JoVE Journal

Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

9.4K vistas.  University of Pennsylvania. Nuevos factores de acogida implicados en la infección viral puede ser identificado a través de celulares basados ​​en el genoma pérdida de la función de detección de RNAi. A Drosophila modelo de cultivo es particularmente susceptible a este enfoque debido a la facilidad y la eficiencia de RNAi. Aquí se demuestra que esta técnica con Vaccinia Virus como un ejemplo.

Video: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Disección de Host-virus Interacción en la replicación lítica de un Herpesvirus Modelo

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Investigación

Inmunología e Infección

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS mediada por delante de detección genética para la identificación de genes implicados en la resistencia no huésped


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodología Para La Generación Eficiente De Proteínas Del Virus Vaccinia Marcadas Fluorescentemente

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Los ensayos basados ​​en la microscopía para la selección de alto rendimiento de los factores del huésped que participan en<em&gt; Brucella</em&gt; Infección de células Hela

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Selección de ensayos para caracterizar nuevos reguladores endoteliales implicados en la respuesta inflamatoria

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Infecciones del arbovirus como herramientas de detección para la identificación de factores antivirales inmunomoduladores y anfitrión Viral

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Proyección de imagen de interacción de células en Mucosa traqueal durante la infección del Virus de la Influenza usando microscopia Intravital de dos fotones

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Establecimiento de la infección Viral y análisis de la interacción huésped-Virus en Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...