Whitehead Institute Fellows Fonds

Partie 1: Mise en place de l&#39;infection <ol><li> Cultiver des cellules HeLa dans des flacons et attendre que les cellules sont d&#39;environ 80% de confluence. </li><li> Préparez assez moyennes croissance virale de l&#39;expérience: régulière DMEM avec 2% de FBS et sans antibiotiques ajoutés. </li><li> L&#39;infection peut être réalisée soit avec un stock brut de virus de la vaccine avec un titre connu, ou saccharose virus purifié avec un titre connu, si vous êtes préoccupé par l&#39;expression du gène hôte. </li><li> Si vous utilisez le virus du saccharose purifié, passez directement à la partie 2. </li><li> Si vous utilisez un stock de virus vaccine brut, juste avant l&#39;infection, soniquer une aliquote du virus dans un sonicateur tasse avec un bain de glace et la plupart du temps un peu d&#39;eau. <ul style="list-style-type:lower-alpha;"><li> Soniquer à environ 30W pendant 20 secondes. </li><li> Vortexer le tube et répétez la sonication trois fois, en ajoutant plus de glace si nécessaire pour garder la tasse remplie de glace et de froid. </li><li> Vortex entre chaque étape de sonication. </li><li> Passez à la partie 2. </li></ul></li><li> Alternativement, si vous ne possédez pas un sonicateur tasse, mélangez un volume égal du stock de virus brut et la trypsine 0.25mg/mL. <ul style="list-style-type:lower-alpha;"><li> Vortex vigoureusement. </li><li> Incuber le stock de virus / trypsine mélanger dans un bain-marie à 37 ° C pendant 30 minutes. </li><li> Vortex à intervalles de 5-10 minutes. </li><li> Passez à la partie 2. </li></ul></li></ol> Partie 2: infecter les cellules <ol><li> Calculer la quantité de particules virales nécessaires pour infecter la monocouche à la multiplicité désirée d&#39;infection (MOI). Typiquement, un MOI élevé (entre 5 et 10) est utilisé pour timecourses infection. </li><li> Ajouter la quantité voulue de virus de saccharose purifié ou trypsinisées virus de brut à 37 ° C les milieux de croissance virale et bien mélanger. Vous devez utiliser les médias suffit pas de couvrir le fond de la fiole. Par exemple, 10 ml de médias pour un flacon T-175. </li><li> Retirez le support à partir des cellules et rincer à température ambiante PBS. </li><li> Ajouter le virus / media croissance virale dans chaque flacon. <ul style="list-style-type:lower-alpha;"><li> Agiter doucement, assiettes d&#39;inclinaison et incuber à 37 ° C dans un incubateur à 5% de CO2 pendant 1 heure. </li><li> Plaques Tilt et agiter toutes les 15 minutes pour la propagation du virus de façon uniforme et garder les cellules humides. </li><li> Pour un point de temps 0hr/Mock, ajouter le support de croissance virale uniquement, sans virus ajouté. </li></ul></li><li> Après l&#39;incubation de 1 heure, retirer le support contenant le virus, et rincer trois fois avec la température ambiante du PBS. </li><li> Ajouter le montant maximum du milieu de croissance virale dans chaque flacon. Par exemple, 30 ml de médias pour un flacon T-175. Commencez à compter ce que votre point 0 h de temps. </li></ol> Partie 3: Récolte des cellules <ol><li> Vérifiez les cellules sous un microscope et de noter tout effet cytopathogène (ECP). </li><li> Retirez le support et rincer les cellules avec 30 ml de la température ambiante du PBS. </li><li> Ajouter 15 ml de trypsine dans les cellules et incuber pendant 2-5min à 37 ° C. </li><li> Vérifiez sous le microscope pour le détachement des cellules et appuyez délicatement flacon pour déloger les cellules. </li><li> Transfert cellules avec une pipette stérile sérologique dans un tube conique de 50 ml. </li><li> Laver le ballon avec un volume égal de milieu de croissance cellulaire, et d&#39;ajouter le support pour les cellules traitées à la trypsine dans le tube conique. </li><li> Centrifuger les cellules à 300 x g pendant 5 minutes à température ambiante. </li><li> Retirez la trypsine / media à partir du culot cellulaire. </li><li> Reprendre le culot cellulaire soit réactif Trizol ou le tampon de lyse à partir de votre kit d&#39;isolement désiré ARN. </li><li> Si vous utilisez TRIzol, diviser l&#39;échantillon en aliquotes de 1 mL dans des tubes Eppendorf 1,5 ml étiquetés et congeler à -80 ° C. </li><li> Répétez l&#39;opération pour tous les points du temps, un flacon de récolte par point de temps. </li></ol> Partie 4: extraction d&#39;ARN des échantillons dans TRIzol <ol><li> À ce stade, vos échantillons doivent être remis en suspension dans du réactif Trizol et prêtes à être traitées. </li><li> Ajouter 200 ul de réactif PCA séparation de phase pour chaque 1 mL de Trizol dans chaque tube. Vous pouvez avoir besoin de transférer l&#39;échantillon à un plus grand tube, si vous êtes débutant avec une grande quantité de TRIzol. </li><li> Vortex ou secouer vigoureusement et incuber à température ambiante pendant 2-3 minutes. </li><li> Centrifuger l&#39;échantillon à 12 000 xg pendant 15 minutes. </li><li> Transférer la phase aqueuse dans un nouveau tube. La phase aqueuse est la couche claire sur le dessus. Vous devriez être en récupérer environ 600μL pour chaque 1 ml de Trizol vous avez commencé avec. </li><li> Faites une deuxième extraction phénol / chloroforme, en utilisant cette fois 500μL de chloroforme pour 1 ml TRIzol, au lieu de la BCP. </li><li> Vortex ou secouer vigoureusement et incuber à température ambiante pendant 2-3 minutes. </li><li> Centrifuger l&#39;échantillon à 12 000 xg pendant 15 minutes. </li><li> Transférer la phase aqueuse dans un nouveau tube. La phase aqueuse est la couche claire sur le dessus. </li><li> Ajouter 20 pg d&#39;acrylamide linéaire pour chaque échantillon. L&#39;acrylamide linéaire agit comme un transporteur et contribue à précipiter l&#39;ARN. </li><li> Ajouter 500 &amp; MiCRO; L d&#39;isopropanol pour 1 ml de Trizol vous avez commencé avec à chaque échantillon et bien mélanger. </li><li> Incuber les échantillons à température ambiante pendant 10 minutes. </li><li> Centrifuger dans une microcentrifugeuse à vitesse maximale pendant 10-15 minutes. </li><li> Le culot d&#39;ARN sera visible au fond du tube. Très soigneusement, éliminer le surnageant du tube, soit avec un aspirateur, ou manuellement par pipette. Ne pas perturber le culot. </li><li> Laver le culot avec 1 ml d&#39;éthanol à 70%. <ul style="list-style-type:lower-alpha;"><li> Ajouter 1 ml d&#39;éthanol à 70% au culot. </li><li> Centrifuger à vitesse maximale 14 000 g pendant 5-7 minutes. </li><li> Très soigneusement, éliminer l&#39;éthanol à partir du tube, soit avec un aspirateur, ou manuellement par pipette. Vérifiez que la pastille est visible au fond du tube. </li><li> Jeter le surnageant. </li></ul></li><li> Répéter l&#39;étape de lavage. Après le surnageant est éliminé, marquer l&#39;endroit où culot est sur le tube. </li><li> Air culot sec pour pas plus de 5 minutes. Ne pas trop sécher, ou l&#39;ARN sera difficile à reconstituer! </li><li> Si vous souhaitez suivre le traitement optionnel DNase pour éliminer l&#39;ADN restant de l&#39;échantillon, resuspendre le culot dans 17μL d&#39;eau sans nucléase. Sinon, resuspendre le culot dans 20 pi d&#39;eau sans nucléase et continuer à l&#39;étape 22. </li><li> (Facultatif traitement à la DNase) Utilisation du Qiagen RNase-free DNase jeu, ajoutez 2μL tampon et 1 microlitre RDD reconstitué sans RNase DNase I de l&#39;ARN en suspension. Mélanger délicatement. </li><li> (Facultatif traitement à la DNase) Incuber à 37 ° C pendant 30 minutes. </li><li> (Facultatif traitement à la DNase) Ajouter 2μL de 2,5 mM d&#39;EDTA et incuber à 65 ° C pendant 5 minutes pour inactiver la DNase. Ne pas dépasser le temps d&#39;inactivation ou la température que plus les temps / températures élevées peut entraîner la dégradation de l&#39;ARN. </li><li> Vérifiez la concentration en ARN par spectrophotomètre. </li><li> Conserver l&#39;échantillon d&#39;ARN à -80 ° C. </li><li> (QC Facultatif) Vérifiez la qualité de l&#39;ARN en utilisant une BioAnalyzer Agilent, ou en exécutant l&#39;échantillon sur un gel dénaturant et en vérifiant les lectures de DO. (260 nm et 260/280 ratio) </li></ol>

<ol>
	<li>KH, R. u. b. i. n. s., LE, H. e. n. s. l. e. y., GW, B. e. l. l., Wang, C., EJ, L. e. f. k. o. w. i. t. z., PO, B. r. o. w. n., DA, R. e. l. m. a. n. <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., JM, G. a. t. e. l. l., 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>

Étapes critiques Partie 1 &amp; 2 Il ya plusieurs étapes essentielles à la mise en place d&#39;une infection synchrone de la vaccine, le premier en faisant attention de sonication (ou trypzinizing) du virus, afin de désagréger les particules virales. Vaccine est fortement sujette à l&#39;agrégation, et la perturbation des particules de virus est importante pour assurer même l&#39;infection de cellules. Afin d&#39;atteindre une infection synchrone, une MOI éle...

<!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>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>Similar reagents, such as TriPure from Roche, will also work.</td>
</tr>
<tr>
<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>Reagent</td>
<td>Qiagen</td>
<td>79254</td>
<td>DNase treatment is an optional step.</td>
</tr>		</tbody>
		</table>
		</div>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

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 1 at JoVE.com

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

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 1

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

10.2K 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 1

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 2

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 2 of 3.

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

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

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 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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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 ...