Nos gustaría dar las gracias a Abbie Spinner en el Centro Nacional de Investigación de Primates de California por su ayuda con la citometría de flujo. Este estudio fue apoyado por fondos del Instituto Nacional de Alergias y Enfermedades Infecciosas (NIAID), Institutos Nacionales de Salud (NIH) AI073745, AI080799 y AI078183.

<ol><li> Antes de iniciar el protocolo, es importante para determinar que las células de mosquito utilizados en el experimento son saludables y en la confluencia ~ 80% si el tratamiento (Figura 1). Células cubierto o insalubres se traducirá en la eficiencia de transfección de baja y le dará los resultados inconsistentes. </li><li> Calentar todos los medios de comunicación antes de su uso en un baño de agua de 28 ° C durante 30 minutos. <ol class="lalpha"><li> A. stephensi MSQ43 células requieren E5 medio: MEM, w Earle / glutamina, 5% de FCS inactivado por calor, el 0,2% de D-glucosa, 1% de penicilina-estreptomicina, y el 1% no aminoácidos esenciales. </li><li> A. gambiae células SUA5B requieren S2 medio: Drosophila de Schneider medio, el 5% de FCS inactivado por calor, y el 1% de penicilina-estreptomicina antibiótico </li></ol></li><li> Descongelar y almacenar todos los materiales de reactivo de transfección, plásmidos y oligonucleótidos en el hielo. </li><li> Todos los procedimientos deben llevarse a cabo en una campana de cultivo de tejidos utilizando una técnica estéril. </li><li> Para células adherentes (A. gambiae SUA5B, A. stephensi MSQ43) aspirado de los viejos medios sin molestar a las células, añadir un volumen equivalente de medio fresco que se ha calentado a 28 ° C, y luego enjuague las células de la parte inferior de la frasco con una pipeta. </li><li> La concentración de células debe ser determinada y ajustada a una concentración final de 1x10 6 células / ml. Las células se pueden mantener a temperatura ambiente, mientras que la preparación de materiales de transfección reactivo. </li><li> Añadir 1 ml de células de mosquito (1x10 6 células / ml) a cada pocillo de una placa de 6 pocillos. Establecer un plato para cada condición experimental (ver más abajo). La tabla I es un sistema de pipeteo para una transfección general. La Tabla II resume cinco condiciones para la prueba de mutagénesis con éxito de un solo nucleótido con BFP específicos LNA-ons. Los dos primeros, y pGFP pBFP, son los controles positivos y negativos, respectivamente. El resto son condiciones experimentales de pBFPs con concentraciones crecientes de BFP específicos LNA-ons. En los pasos 8 a 12, añadir un volumen adecuado de reactivo en consecuencia para cada condición. </li><li> La transferencia de ADN plásmido a un tubo de microcentrífuga estéril de 1,5 ml y agregar el volumen apropiado de tampón de la CE. <ol class="lalpha"><li> Para un ADN plásmido l (1 mg / l), añadir 99 l de tampón CE. </li><li> Por un pGFP l (1 g / l), añadir 99 l de tampón CE. </li><li> Por un pBFP l (1 g / l), añadir 99 l de tampón CE. </li><li> Por un pBFP l (1 g / l) y una l ON (mg / l), añadir 98 l de tampón CE. </li><li> Por un pBFP l (1 g / l) y 5 l ON (1 g / l), añadir 94 l de tampón CE. </li><li> Por un pBFP l (1 g / l) y 10 l ON (1 g / l), añadir 89 l de tampón CE. </li></ol></li><li> Luego agregue lentamente reforzador por 1 mg de ADN utilizado. <ol class="lalpha"><li> Por un plásmido de ADN l, añadir 8 l Enhancer. </li><li> Por un pGFP l, añadir 8 l Enhancer. </li><li> Por un pBFP l, añadir 8 l Enhancer. </li><li> Por un pBFP l y l 1 ON, añadir 16 l Enhancer. </li><li> Por un pBFP l y l 5 ON, añadir 48 l Enhancer. </li><li> Por un pBFP l l 10 y ON, añadir 88 l Enhancer. </li></ol></li><li> Vortex ADN / buffer / Enhancer mezcla durante 1 segundo y asegúrese de que toda la solución está en la parte inferior del tubo. A continuación, se incuba a temperatura ambiente durante 5 min. </li><li> Añadir el reactivo Effectene por ADN plásmido 1 mg y agite por 10 segundos. Compruebe que toda la solución está en el fondo del tubo y luego se incuban a temperatura ambiente durante 10 min. <ol class="lalpha"><li> Por un plásmido de ADN l, añadir 10 Effectene l (Tabla I). </li><li> Por un pGFP l, añadir 10 Effectene l (Tabla II). </li><li> Por un pBFP l, añadir 10 Effectene l (Tabla II). </li><li> Por un pBFP l y l 1 ON, añadir 20 Effectene l (Tabla II). </li><li> Por un pBFP l y l 5 ON, añadir 60 Effectene l (Tabla II). </li><li> Por un pBFP l l 10 y ON, añadir 110 Effectene l (Tabla II). </li></ol></li><li> Llevar a cada tubo a un volumen final de 1000 l mediante la adición de los medios de comunicación calentado gota a gota a la mezcla de ADN / buffer / Enhancer / Effectene. ADN puede precipitar en este momento si los medios de comunicación se añade demasiado rápido. Mezclar suavemente con la pipeta hacia arriba y abajo 3-4 veces. <ol class="lalpha"><li> Por un plásmido de ADN l, añadir los medios de comunicación 882 l (tabla I). </li><li> Por un pGFP l, añadir los medios de comunicación 882 l (Tabla II). </li><li> Por un pBFP l, añadir los medios de comunicación 882 l (Tabla II). </li><li> Por un pBFP l y l 1 ON, añadir 864 medios de comunicación l (Tabla II). </li><li> Por un pBFP l y l 5 ON, añadir 792 medios de comunicación l (Tabla II). </li><li> Por un pBFP l l 10 y ON, añadir los medios de comunicación 702 l (Tabla II). </li></ol></li><li> A continuación se añade lentamente 1 ml de la correspondiente ADN / Enhancer / Effectene mezcla a cada pocillo una gota a la vez. </li><li> Agitar la placa suavemente para asegurar la distribución uniforme de las células y la mezcla de transfección. Incubatlas células e bajo condiciones normales de crecimiento. </li><li> Un día después de la transfección, suavemente aspire a sustituir a los medios de comunicación y poco a poco, con un volumen equivalente de los medios de comunicación recién calentado. Es importante no perturbar las capas de células en la parte inferior de los pozos cuando se hace esto. </li><li> Dos días después de la transfección examinar las células positivas de control transfectadas con pGFP bajo microscopio de fluorescencia para determinar la eficiencia de transfección (Figura 2). </li><li> SUA5B células crecen muy rápidamente y se convertirá en maleza. Cuatro días después de la transfección de estas células delgadas aspirando a los medios de comunicación antiguos y agregar poco a poco un volumen equivalente de los medios de comunicación recién calentado. </li><li> Después de la adición de nuevos medios de comunicación a los pozos, desmonte todas las células de la parte inferior de cada pozo pipeta de arriba a abajo, descartar la mitad del volumen (1 ml) de la mezcla de células y agregar 1 ml de agua dulce caliente medios para llevar el volumen final a 2 mL. </li><li> Revisar las células diariamente para asegurarse de que están sanos y no cubierto, fina como sea necesario. </li><li> Permiten que las células crezcan en condiciones normales de cinco a siete días antes de la transfección a su recogida para su análisis. </li><li> De cinco a siete días después de la transfección, recoger las células mediante la aspiración de los viejos medios en todos los pozos y la adición de 1 ml de PBS estéril a temperatura ambiente a cada pocillo. </li><li> Separar las células de la parte inferior del pozo pipeteando arriba y hacia abajo y la transferencia de mezcla de células de 5 tubos con tapa de presión ml y de inmediato analizar las células mediante citometría de flujo. </li></ol> Resultados representante <img src="/files/ftp_upload/2355/2355fig1.jpg" alt="Figura 1" /> Figura 1. A. untransfected saludable stephensi MSQ43 (A) y A. gambiae SUA5B (B) las células en la confluencia de aproximadamente 80%. <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="Figura 2" /> Figura 2. A. stephensi MSQ43 (A) y A. gambiae SUA5B (B) las células transfectadas con GFP control positivo expresar plásmido. <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="Figura 3" /> Figura 3. Citometría de flujo de datos que confirma la conversión de BFP a las buenas prácticas agrarias a través de la mutagénesis de un solo nucleótido siguientes transfección con BFP específicas de ácido nucleico cerrado-oligonucleótidos modificados (LNA-ons). SUA5B células fueron transfectadas con un plásmido que expresa GFP (pGFP) como control positivo, con un plásmido que expresa BFP (pBFP) como control negativo, o con pBFP y concentraciones crecientes de BFP específicos LNA-ons. (A) de apertura de puerta estrategia para los datos de citometría de flujo que muestra de izquierda a derecha: dispersión frontal contra lado de las células vivas, yoduro de propidio (PI), las células negativas, y las células GFP positivos cinco días después de la transfección. (B) Gráfico de porcentaje de células positivas GFP se muestra en (A) transfección siguientes pBFP y concentraciones crecientes de LNA-ons. <img src="/files/ftp_upload/2355/2355table1.jpg" alt="Tabla 1" /> Tabla I. condiciones de transfección. <img src="/files/ftp_upload/2355/2355table2.jpg" alt="Tabla 2" /> Tabla II. Condiciones de transfección utilizado en la Figura 1.

<ol>
	<li>Corby-Harris, V. D. A., Watkins de Jong, L., Pakpour, N., Antonova, Y., Ziegler, R., Ramberg, F., Lewis, E. E., Brown, J. M., Luckhart, S. L., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+controlling+malaria+transmission+in+the+mosquito+Anopheles+stephensi.">A novel strategy for controlling malaria transmission in the mosquito Anopheles stephensi.</a> PLOS Pathogens. , (2010).</li><li>Braasch, D. A., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid+(LNA):+fine-tuning+the+recognition+of+DNA+and+RNA.">Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.</a> Chem Biol. 8, 1-7 (2001).</li><li>Andrieu-Soler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transmission+of+targeted+gene+modification+using+single-stranded+oligonucleotides+with+flanking+LNAs.">Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.</a> Nucleic Acids Res. 33, 3733-3742 (2005).</li><li>Parekh-Olmedo, H., Drury, M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nucleotide+exchange+in+Saccharomyces+cerevisiae+directed+by+short+oligonucleotides+containing+locked+nucleic+acids.">Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids.</a> Chem Biol. 9, 1073-1084 (2002).</li><li>Rhee, W. J., Santangelo, P. J., Jo, H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+accessibility+and+signal+specificity+in+live-cell+detection+of+BMP-4+mRNA+using+molecular+beacons.">Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.</a> Nucleic Acids Res. 36, e30-e30 (2008).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+SNPs+with+molecular+beacons.">Genotyping SNPs with molecular beacons.</a> Methods Mol Biol. 212, 111-128 (2003).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat Biotechnol. 16, 49-53 (1998).</li><li>Blandin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+genetic+basis+of+resistance+to+malaria+parasites+in+Anopheles+gambiae.">Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.</a> Science. 326, 147-150 (2009).</li><li>Niare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+loci+affecting+resistance+to+human+malaria+parasites+in+a+West+African+mosquito+vector+population.">Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.</a> Science. 298, 213-216 (2002).</li><li>Riehle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+malaria+infection+in+Anopheles+gambiae+is+regulated+by+a+single+genomic+control+region.">Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.</a> Science. 312, 577-579 (2006).</li></ol>

Aquí presentamos un método in vitro y las pruebas para la conversión selectiva de un solo nucleótido en el gen episomal un objetivo después de la transfección de LNA-Ons en A. stephensi y A. gambiae células. LNA-ON mediada por la conversión de genes requiere la inducción de un sitio específico de la respuesta de reparación del ADN, nuestros datos indican que las células de mosquito puede apoyar este mecanismo de mutagénesis sitio-específica. Mientras que el método que a...

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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>Effectene transfection reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K4935-00</td>
<td>&nbsp;</td>
<tr>
<td>BFP plasmid (pcDNA5/FRT/TO)</td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LNA-ONs </td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM, Earle's w/ glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s Drosophila medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11730</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>10% D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin antibiotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides

Parásitos Plasmodium, el agente causante de la malaria, se transmite por la picadura de mosquitos anofeles infectados que resulta en más de 250 millones de nuevas infecciones cada año. A pesar de décadas de investigación, aún no existe una vacuna contra la malaria, destacando la necesidad de estrategias de control de la novela. Un enfoque innovador es el uso de mosquitos genéticamente modificados para controlar de forma efectiva la transmisión de parásitos de malaria. Alteraciones deliberadas de vías de señalización celular en el mosquito, a través de mutagénesis dirigida, se han encontrado para regular el desarrollo del parásito 1. A partir de estos estudios, podemos empezar a identificar posibles objetivos para la transformación genética. Mutagénesis dirigida se ha basado tradicionalmente en la recombinación homóloga entre un gen diana y una molécula de ADN de gran tamaño. Sin embargo, la construcción y el uso de tales moléculas de ADN complejo para la generación de líneas celulares transformadas de forma estable es costoso, lento y con frecuencia ineficientes. Por lo tanto, una estrategia de uso de ácido nucleico cerrado modificado-oligonucleótidos (LNA-ons) ofrece una alternativa útil para la introducción artificial sustituciones de un solo nucleótido en el gen episomal objetivos y ADN cromosómico (revisado en 2). LNA-ON mediada por mutagénesis dirigida se ha utilizado para introducir mutaciones puntuales en genes de interés en cultivos de células de levadura y 3,4 ratones. Mostramos aquí que LNA complementos pueden ser utilizados para introducir un cambio de nucleótido único en un objetivo episomal transfectadas que se traduce en un cambio de la proteína fluorescente azul (BFP) expresión de la proteína verde fluorescente (GFP) de expresión, tanto en Anopheles gambiae y Anopheles stephensi células . Esta conversión se demuestra por primera vez que la mutagénesis eficaz de los genes diana en las células de mosquito puede estar mediado por LNA-ons y sugiere que esta técnica puede ser aplicable a la mutagénesis de los objetivos cromosómicas in vitro e in vivo.

Watch this Scientific Journal Video about Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides at JoVE.com

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Oligonucleótidos se puede utilizar para sustituir a un sitio concreto de un solo nucleótido de los genes diana transfectadas en ambos<em&gt; Anopheles gambiae</em&gt; Y<em&gt; Anopheles stephensi</em&gt; Células.

Transfección y mutagénesis de los genes diana en células de mosquito por nucleico cerrado modificado ácido oligonucleótidos

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

transfection-mutagenesis-target-genes-mosquito-cells-locked-nucleic

Research

JoVE Journal

Immunology and Infection

10.3K vistas.  University of California, Davis. Oligonucleótidos se puede utilizar para sustituir a un sitio concreto de un solo nucleótido de los genes diana transfectadas en ambos Anopheles gambiae Y Anopheles stephensi Células.

Video: Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

La identificación de genes inducida por la desregulación de Sarcoma de Kaposi asociado al virus del herpes (HVSK)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Investigación

Inmunología e Infección

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antígeno Específico de la En Vivo Matar ensayo utilizando CFSE células diana marcadas

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparación y uso de VIH-1 en células CD4 infectadas Primaria + T-células como células diana en las células asesinas naturales ensayos citotóxicos

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocolo optimizado para la transfección eficiente de células dendríticas, sin maduración de las células

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

La evaluación de la hipermutación somática en Ramos células B después de sobreexpresión o derribo de genes específicos

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Flujo de ácido nucleico cerrado Citometría de fluorescencia In situ</emHibridación> (LNA flujo-FISH): un método para la detección de bacterias pequeñas de ARN

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Identificación de nuevos genes asociados con alginato producción en<em&gt; Pseudomonas aeruginosa</em&gt; Uso de Mini-<em&gt; Himar1</em&gt; La mutagénesis mediada por transposón Mariner

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.