Whitehead Institute Fellows Fonds

Partie 1: synthèse de l&#39;ADNc de l&#39;ARN <ol><li> Avant d&#39;utiliser le Ambion Amino Allyl MessageAmp II kit, ajouter la quantité recommandée d&#39;éthanol à 100% pour les tampons de lavage. </li><li> Dans le tube de réaction PCR, ajouter entre 100 ng d&#39;ARN total de 5 ug et 1 microlitre de T7 oligo (dT) primer. Porter le volume à 12μl avec une eau sans nucléase. </li><li> Incuber les échantillons à 70 ° C pendant 10 min dans un thermocycleur. </li><li> Retirer les échantillons d&#39;ARN à partir de 70 ° C et centrifuger brièvement. Placez-le sur la glace. </li><li> Préparer le 1 er volet de synthèse master mix et de conserver à température ambiante (avec excédent de 10% pour le pipetage d&#39;erreur). (Tableau 1) </li><li> Mélanger délicatement la pipette ou feuilleter Maître de mélanger, puis centrifuger brièvement. </li><li> Transfert de l&#39;8μl Master Mix pour chaque échantillon d&#39;ARN. Mélanger par aspiration et refoulement 3-4 fois. </li><li> Incuber à 42 ° C pendant 2 heures dans un thermocycleur. </li><li> Centrifuger les échantillons brièvement et placer sur la glace. Procéder immédiatement à l&#39;étape suivante de la synthèse des ADN double brin. </li><li> Préparer le 2 ème volet maîtrise de synthèse mélange sur la glace (avec excédent de 10% pour le pipetage d&#39;erreur). (Tableau 2) </li><li> Mélanger délicatement la pipette ou feuilleter Maître de mélanger, puis centrifuger brièvement. </li><li> Transfert 80μl à chaque échantillon et mélanger délicatement par aspiration et refoulement 3-4 fois. </li><li> Incuber à 16 ° C pendant 2 heures dans un thermocycleur. </li><li> Après l&#39;incubation de 2 heures, procéder à l&#39;ADNc de nettoyage étape ou congeler immédiatement à -20 ° C. </li></ol> Partie 2: l&#39;ADNc double brin de nettoyage <ol><li> Retirez la pure ADNc du réfrigérateur et le laisser se stabiliser à la température ambiante pendant 30 minutes avant utilisation. Agitez la bouteille pour bien remettre en suspension les perles magnétiques ADNc contraignante avant l&#39;utilisation. </li><li> Aliquoter eau sans nucléase dans un tube de 1,5 ml et incuber à 50-60 ° C pendant au moins 10 minutes pendant l&#39;incubation précédentes 2hr. </li><li> Ajouter 180μl de Pure ADNc de chaque échantillon, et bien mélanger par pipetage de haut en bas. Transférer les échantillons à un 96-même à fond rond assiette. </li><li> Continuer à mélanger les échantillons en agitant la plaque sur un agitateur orbital pendant au moins 2 minutes. </li><li> Déplacer la plaque à un support magnétique pour capturer les billes magnétiques. Laisser la plaque sur le stand pendant environ 6 minutes ou jusqu&#39;à ce que le mélange devient transparent et les perles à caractère contraignant ont granulés. </li><li> Aspirer délicatement le surnageant avec un aspirateur sans déranger les billes magnétiques. Sinon, retirez délicatement le surnageant avec une pipette et jeter le surnageant. </li><li> Retirer la plaque du socle magnétique. </li><li> Ajouter 150μl de tampon de lavage d&#39;ADNc dans chaque puits et agiter la plaque pendant 1 minute sur l&#39;agitateur orbital à vitesse modérée. Perles va se dispersent pas à cette étape, en raison de la faible tension de surface du tampon de lavage. </li><li> Déplacer la plaque à un support magnétique pour capturer les billes magnétiques. </li><li> Aspirer délicatement le surnageant avec un aspirateur sans déranger les billes magnétiques. Sinon, retirez délicatement le surnageant avec une pipette et jeter le surnageant. </li><li> Retirer la plaque du socle magnétique. </li><li> Répéter le lavage une 2 ème fois avec 150μl de tampon de lavage d&#39;ADNc. </li><li> Après le lavage 2 ème, sécher les perles en secouant la plaque pendant 2 minutes sur l&#39;agitateur orbital à la vitesse maximale. Ne pas trop sécher! </li><li> Éluer l&#39;ADNc à partir des billes en ajoutant 18μl de l&#39;préchauffé eau sans nucléase à chaque échantillon. </li><li> Secouez vigoureusement la plaque pendant 3 minutes sur l&#39;agitateur orbital, puis vérifiez que les billes magnétiques sont totalement dispersés. Sinon, continuez secouant. </li><li> Une fois que les billes magnétiques ont complètement dispersé, déplacer la plaque d&#39;un support magnétique pour capturer les billes magnétiques. </li><li> Soigneusement transfert de l&#39;ADNc élué (~ 16μl) d&#39;une nouvelle plaque PCR (ou tubes PCR). </li><li> Passez directement à l&#39;étape suivante, ou geler l&#39;ADNc à -20 ° C. </li></ol> Partie 3: transcription in vitro (IVT) <ol><li> Préparer le mélange maître IVT à température ambiante. (Tableau 3) </li><li> Délicatement la pipette du Master Mix ou à cran de se mélanger, et centrifuger brièvement. </li><li> Ajouter 24μl de chaque échantillon, et mélanger délicatement par aspiration et refoulement 3-4 fois. </li><li> Incuber à 37 ° C pendant 14 heures dans un thermocycleur, puis maintenez à 4 ° C jusqu&#39;au moment de l&#39;étape suivante. </li></ol> Partie 4: ARNa nettoyage après IVT <ol><li> Vortex les perles liaison à l&#39;ARN brièvement pour obtenir un mélange homogène avant utilisation. </li><li> Préparer le ARNa Mix reliure à température ambiante (tableau 4) Cela peut être fait à l&#39;avance -. Le mélange préparé contraignantes peuvent être stockés à température ambiante pendant jusqu&#39;à une semaine. </li><li> Mélangez bien au vortex. </li><li> Aliquoter le tampon d&#39;élution ARNa dans un tube de 1,5 ml et incuber à 50-60 ° C pendant au moins 10 minutes. </li><li> Ajouter 70μl de l&#39;ARNa mélange de liantà chaque échantillon et bien mélanger par pipetage haut et en bas de 3-4 fois. </li><li> Transférer les échantillons de la plaque PCR de 96 puits à fond rond assiette. </li><li> Ajouter 50 pl de 100% d&#39;isopropanol à chaque échantillon et bien mélanger par pipetage haut et en bas de 3-4 fois. </li><li> Secouez doucement la plaque sur un agitateur orbital pendant au moins 2 minutes pour bien mélanger les échantillons. </li><li> Déplacer la plaque à un support magnétique pour capturer les billes magnétiques. Laisser la plaque sur le stand jusqu&#39;à ce que le mélange devient transparent et les perles à caractère contraignant ont granulés. </li><li> Aspirer délicatement le surnageant avec un aspirateur sans déranger les billes magnétiques. Sinon, retirez délicatement le surnageant avec une pipette et jeter le surnageant. </li><li> Retirer la plaque du socle magnétique. </li><li> Ajouter 100 ul Solution Laver ARNa dans chaque puits et agiter la plaque pendant 1 minute sur l&#39;agitateur orbital à vitesse modérée. Perles peut pas entièrement se dispersent à cette étape. </li><li> Déplacer la plaque à un support magnétique pour capturer les billes magnétiques. </li><li> Aspirer délicatement le surnageant avec un aspirateur sans déranger les billes magnétiques. Sinon, retirez délicatement le surnageant avec une pipette et jeter le surnageant. </li><li> Retirer la plaque du socle magnétique. </li><li> Répétez le laver une fois 2 ème avec 100 ul Solution Laver ARNa. </li><li> Après le lavage 2 ème, sécher les perles en secouant la plaque pendant 1 minute sur l&#39;agitateur orbital à la vitesse maximale. Ne pas trop sécher les échantillons! </li><li> Eluer la ARNa des billes en ajoutant 40μl de tampon d&#39;élution préchauffé ARNa à chaque échantillon. </li><li> Secouez vigoureusement la plaque sur l&#39;agitateur orbital pendant 3 minutes, puis vérifiez que les billes magnétiques sont totalement dispersés. Sinon, continuez secouant. </li><li> Une fois que les billes magnétiques ont complètement dispersé, déplacer la plaque d&#39;un support magnétique pour capturer les billes magnétiques. Le surnageant contient les ARNa nettoyé aminés allyle incorporé. </li><li> Soigneusement transférer le ARNa élué à une nouvelle plaque PCR (ou tubes PCR). </li><li> (Étape facultative) Vérifiez la concentration en ARN des échantillons en mesurant 1.5μl sur un spectrophotomètre NanoDrop. </li><li> Immédiatement continuer sur la prochaine étape, ou de stocker l&#39;ARNa à -80 ° C. </li></ol> Tableau 1: Volet 1 er ADNc Synthèse Mix Master <table border="1" cellpadding="8"><tbody><tr><td> Réactifs </td><td> Montant pour une réaction </td></tr><tr><td> Tampon 10X First Strand </td><td> 2 pl </td></tr><tr><td> dNTP Mix </td><td> 4 pl </td></tr><tr><td> Inhibiteur de la ribonucléase </td><td> 1 pl </td></tr><tr><td> Script tableau </td><td> 1 pl </td></tr></tbody></table> Tableau 2: ADNc 2 ème volet Maître Synthèse Mix <table border="1" cellpadding="8"><tbody><tr><td> Réactifs </td><td> Montant pour une réaction </td></tr><tr><td> Eau sans nucléase </td><td> 63 ul </td></tr><tr><td> Tampon 10X deuxième brin </td><td> 10 ul </td></tr><tr><td> dNTP Mix </td><td> 4 pl </td></tr><tr><td> ADN polymérase </td><td> 2 pl </td></tr><tr><td> RNase H </td><td> 1 pl </td></tr></tbody></table> Tableau 3: IVT Mix Master <table border="1" cellpadding="8"><tbody><tr><td> Réactifs </td><td> Montant pour une réaction </td></tr><tr><td> aaUTP solution (75 mM) </td><td> 2 pl </td></tr><tr><td> ATP, CTP, GTP mélange (25 mM) </td><td> 12 ul </td></tr><tr><td> T7 UTP solution (75 mM) </td><td> 2 pl </td></tr><tr><td> Tampon de réaction 10X T7 </td><td> 4 pl </td></tr><tr><td> T7 Enzyme Mix </td><td> 4 pl </td></tr></tbody></table> Tableau 4: Mélanger ARNa reliure <table border="1" cellpadding="8"><tbody><tr><td> Réactifs </td><td> Montant pour une réaction </td></tr><tr><td> Perles liaison à l&#39;ARN * </td><td> 10 ul </td></tr><tr><td> * Remise en suspension de billes Solution </td><td> 4 pl </td></tr><tr><td> 100% d&#39;isopropanol ** </td><td> 6 pl </td></tr><tr><td> Concentré ARNa Binding Buffer </td><td> 50 ul </td></tr></tbody></table> * Mélanger les perles liaison à l&#39;ARN avec la solution resuspension premier talon. ** Ajouter l&#39;isopropanol et bien mélanger avant d&#39;ajouter le concentré de tampon de liaison ARNa.

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, 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.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

The authors have nothing to disclose.

Étapes critiques Un second tour de l&#39;amplification n&#39;est pas conseillé que des biais dans les données array ont été observées. Mélanger soigneusement à chaque étape enzymatique (1 er et 2 ème synthèse brin d&#39;ADNc, IVT) est essentielle à l&#39;obtention de rendements d&#39;amplification bon, tout comme l&#39;incubation de chaque étape enzymatique à la température appropriée. Un cycleur PCR avec couvercle chauffant réglable est préféré - les ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
</tr>		</tbody>
		</table>
		</div>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

La famille<em&gt; Poxviridae</em&gt; Se compose de grandes double-brin d&#39;ADN contenant des virus qui se répliquent exclusivement dans le cytoplasme des cellules infectées. Les membres de la<em&gt; Orthopoxvirus</em&gt; Genre comprennent la variole, l&#39;agent causal de la variole humaine, simienne, et la vaccine (ACC), le membre prototypique de la famille des virus. Dans le génome relativement importante (~ 200 ko) de la vaccine, trois classes de gènes sont encodés: précoce, intermédiaire et tardive. Alors que tous les trois classes sont transcrits par les ARN polymérases virale codée, chaque classe a une fonction différente dans le cycle de vie du virus. Les poxvirus utilisent de multiples stratégies pour la modulation de l&#39;environnement cellulaire de l&#39;hôte pendant l&#39;infection. Afin de comprendre la régulation de l&#39;hôte et l&#39;expression des gènes du virus, nous avons utilisé l&#39;échelle du génome des approches pour analyser l&#39;abondance transcription de deux virus et les cellules hôtes. Ici, nous démontrons infections cours du temps des cellules HeLa par le virus de la vaccine et de l&#39;ARN d&#39;échantillonnage à différents moments après l&#39;infection. Les deux hôtes et virales ARN total est isolé et amplifié pour que l&#39;hybridation des puces pour l&#39;analyse de l&#39;expression génique.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

Protocole pour la vaccine infection de cellules HeLa et l&#39;analyse de l&#39;hôte et l&#39;expression des gènes viraux.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

7.9K vues.  MIT - Massachusetts Institute of Technology. Protocole pour la vaccine infection de cellules HeLa et l'analyse de l'hôte et l'expression des gènes viraux.

Vidéo: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Bacterial Gene Expression Analysis Using Microarrays

Analyse bactérienne expression génique par puces à ADN

Recherche

Biologie

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 1

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 3

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Induite par le virus Gene Silencing (VIGS) dans Nicotiana benthamiana Et aux tomates

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium induite par le virus Assay Gene Silencing En Coton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

La protéine plate-forme de production complexe MultiBac à l&#39;EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...