Les auteurs remercient Matthias Peter et son groupe à l&#39;Institut de biochimie à l&#39;EPF de Zürich où ces méthodes ont été développées. Ce travail a été soutenu par le Fonds national suisse de la science.

Une. Mesures de microscopie <ol><li> Inoculer 5 ml de milieu synthétique avec de la levure. </li><li> Cultiver des cellules à 30 ° C pendant la nuit. </li><li> Mesurer la DO 600 de la culture pendant une nuit, le lendemain matin. </li><li> Diluer culture d&#39;une nuit dans 5 ml de milieu synthétique OD 600 0,05. </li><li> Cultiver la culture diluée à 30 ° C pendant au moins 4 heures. </li><li> Préparer trois fois concentré (0,6 M) une solution de NaCl dans un milieu synthétique. </li><li> Préparer une solution à 1 mg / ml de concanavaline de A (ConA) dans du PBS. </li><li> Filtrat ~ 200 pi de solution ConA dans la glissière de bien. </li><li> Laisser reposer pendant 30 min. </li><li> Retirer la solution ConA. </li><li> Ajouter 150 ul de H 2 O dans le puits. </li><li> Retirer H 2 O. </li><li> Mesurer la DO 600 de la culture cellulaire. </li><li> Préparer 300 pl de culture cellulaire à DO600 = 0,02 dans un microtube de 1,5 ml. </li><li> Soniquer les cellules dans un bain d&#39;eau pendant 45 sec. </li><li> Vortex brièvement la microtube. </li><li> Soniquer les cellules dans un bain d&#39;eau pendant 45 sec. </li><li> Vortex brièvement le microtube. </li><li> Ajouter 200 ul de la culture de cellules dans le puits. </li><li> Attendre 30 minutes pour laisser les cellules se déposent au fond du puits. </li><li> Placer la lame de bien sur le microscope. </li><li> Sélectionnez les réglages de l&#39;éclairage pour le canal de fluorescence à enregistrer. </li><li> Sélectionnez les paramètres d&#39;éclairage pour deux images de fond clair (un peu hors-sujet: 3 um ci-dessous le plan focal pour permettre une segmentation correcte des cellules). </li><li> Sélectionnez les intervalles de temps pour la mesure time-lapse </li><li> Sélectionnez les champs de vue de l&#39;image. </li><li> Lancement de l&#39;acquisition de quelques images. </li><li> Pause de l&#39;acquisition d&#39;ajouter les 100 ul de 0,6 M de NaCl moyen. </li><li> Reprendre imagerie. </li></ol> 2. Les mesures de cytométrie en flux <ol><li> Inoculer 5 ml de milieu synthétique avec de la levure. </li><li> Croître à 30 ° C pendant la nuit. </li><li> Mesurela DO600 de la culture pendant une nuit, le lendemain matin. </li><li> Diluer culture d&#39;une nuit dans 5 ml de milieu synthétique DO 600 de 0,1. </li><li> Croître à 30 ° C pendant au moins 4 heures pour atteindre une DO600 de 0,2-0,4. </li><li> Préparer la solution de cycloheximide 1 mg / ml dans H 2 O. </li><li> Préparer trois fois concentré (0,6 M) une solution de NaCl dans un milieu synthétique. </li><li> Préparer un microtube de 1,5 ml avec 100 ul de 0,6 M de NaCl moyen pour chaque point de temps pour être mesurés. </li><li> Au temps 0, ajouter 200 ul de la culture de cellules à chaque microtube. </li><li> Incuber sous agitation à 30 ° C. </li><li> A l&#39;instant T, ajouter 30 ul de cycloheximide à un microtube. </li><li> Répéter l&#39;étape 2.11 pour tous les points de temps désirés. </li><li> Incuber les microtubes pendant au moins 45 minutes et jusqu&#39;à 3 heures à 30 ° C avec agitation par secousses. </li><li> Préparer des tubes FACS avec 400 ul de PBS. </li><li> Soniquer les cellules dans un bain d&#39;eau pendant 1 min. </li><li> Vortex brièvement les microtubes. </li><li> Filsicat les cellules dans un bain d&#39;eau pendant 1 min. </li><li> Vortex brièvement les microtubes. </li><li> Ajouter 100 ul de culture cellulaire de chaque microtube à son tube FACS correspondant. </li><li> Sélectionnez le laser d&#39;excitation de 488 nm et 530/30 nm filtre passe-bande pour la détection. </li><li> Test de non-expression et exprimer pleinement échantillon pour vérifier si elles tombent dans la plage de sensibilité de détection. </li><li> Mesurer 10.000 cellules pour chaque échantillon acquis. </li></ol>

<ol>
	<li>Gasch, A. P., Spellman, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes.">Genomic expression programs in the response of yeast cells to environmental changes.</a> Molecular biology of the cell. 11 (12), 4241-4257 (2000).</li><li>de Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+Hog1+recruits+Rpd3+histone+deacetylase+to+activate+osmoresponsive+genes.">The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.</a> Nature. 427 (6972), 370-374 (2004).</li><li>Ghaemmaghami, S., Huh, W. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Wu, J. -. Q., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+cytokinesis+proteins+globally+and+locally+in+fission+yeast.">Counting cytokinesis proteins globally and locally in fission yeast.</a> Science. 310 (5746), 310-314 (2005).</li><li>Colman-Lerner, A., Gordon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+cell-to-cell+variation+in+a+cell-fate+decision+system.">Regulated cell-to-cell variation in a cell-fate decision system.</a> Nature. 437 (7059), 699-706 (2005).</li><li>Acar, M., Becskei, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+cellular+memory+by+reducing+stochastic+transitions.">Enhancement of cellular memory by reducing stochastic transitions.</a> Nature. 435 (7039), 228-232 (2005).</li><li>Pelet, S., Rudolf, F., Nadal-Ribelles, M., de Nadal, E., Posas, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+activation+of+the+HOG+MAPK+pathway+regulates+bimodal+gene+expression.">Transient activation of the HOG MAPK pathway regulates bimodal gene expression.</a> Science. 332 (6030), 732-735 (2011).</li><li>de Nadal, E., Casadome, L., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+MEF2-like+transcription+factor+Smp1+by+the+stress-activated+Hog1+mitogen-activated+protein+kinase.">Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase.</a> Molecular and Cellular Biology. 23 (1), 229-237 (2003).</li><li>Elowitz, M. B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome biology. 7 (10), R100 (2006).</li><li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry.">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. Chapter 14, Unit 14.18 (2008).</li><li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images.">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (20), 7165-7170 (2008).</li><li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescence+microscopy+for+systems+biology.">High-throughput fluorescence microscopy for systems biology.</a> Nature reviews Molecular cell biology. 7 (9), 690-696 (2006).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Microscopie Le traitement des puits avec de la ConA est une étape essentielle pour assurer une imagerie appropriée des cellules. Parce que la ConA a une faible solubilité dans du PBS (5 mg / ml), le procédé de filtration permet l&#39;élimination des gros agrégats qui sont présents dans la solution filtrée et interfèrent avec la formation d&#39;image. Les cellules se fixent relativement fortement à la surface traitée et l&#39;addition de la solution ne doit pas perturber l&#39;induct...

La signalisation par l&#39;intermédiaire de cascades de transduction aboutit souvent à l&#39;expression de protéines. La caractérisation de ce profil d&#39;expression est un élément clé dans la compréhension de la fonction des voies biologiques. L&#39;identification du spectre de régulés à la hausse des protéines et la dynamique de leur activation peut être réalisée par diverses techniques telles que les micro-réseaux, des taches ou des taches Nord Ouest 1-3. Cependant, ces moyens techniques de la réponse d&#39;une population entière de cellules. Pour comprendre la régulation fine de l&#39;expression des protéines, il est souhaitable de recueillir des mesures au niveau de la cellule unique. Idéalement, ces mesures devraient également fournir des données quantitatives se prêtent à développer des modèles mathématiques de la voie sous-jacent. Microscopie et cytométrie en flux deux techniques, qui sont idéalement adaptés à la diffusion de telles mesures quantitatives de cellules individuelles. Le marquage endogène des protéines avec une protéine fluorescente peut be utilisé pour quantifier le niveau d&#39;expression de 4. Cependant, étant donné que l&#39;addition d&#39;un grand fragment fluorescent à l&#39;extrémité de la protéine peut rendre non fonctionnel, il est souvent plus souhaitable de générer reporters d&#39;expression spécifiques basés sur un promoteur commandant l&#39;expression d&#39;une construction fluorescent. Cette protéine rapporteur est exogène au système cellulaire et donc n&#39;influence pas les événements de signalisation qui se déroulent dans la cellule. À la fois la microscopie et la cytométrie en flux ont été largement utilisés dans le domaine de signalisation de la levure. A titre d&#39;exemples, Colman-Lerner et collègues corrélés, dans des cellules individuelles, l&#39;expression de l&#39;accouplement journalistes spécifiques et exprimées de manière constitutive par microscopie à quantifier le bruit dans la levure accouplement transduction du signal cascade 5, Acar et ses collègues ont utilisé la cytométrie en flux pour étudier le réseau de régulation contrôler l&#39;expression des gènes GAL 6. Dans une étude précédente 7, nous avons utilisé un combinatisur ces deux techniques pour étudier la sortie de l&#39;expression de l&#39;osmolarité glycérol élevé (HOG) de la voie dans la levure bourgeonnante. Cette mitogen activated protein kinase (MAPK) est déclenchée par le stress hyper-osmotique. Elle se traduit par l&#39;activation des MAPK Hog1, qui subit une translocation vers le noyau de la cellule pour induire la transcription d&#39;un programme résultant dans l&#39;expression d&#39;approximativement 300 gènes. Pour étudier ce processus, nous avions conçu un journaliste d&#39;expression basé sur le promoteur de STL1 (un gène induit spécifiquement en réponse à l&#39;activité de Hog1 8) entraînant l&#39;expression d&#39;une protéine fluorescente Vénus quadruple (p STL1-qV). La cytométrie en flux des mesures ont découvert la présence de deux populations de cellules au niveau de la tension intermédiaire (0,1 M de NaCl) avec seulement une fraction de la population exprimant le rapporteur fluorescent. Nous avons utilisé la microscopie à approfondir ce comportement et découvert que ce bruit dans l&#39;expression de la protéine a été régie par des facteurs intrinsèques 9. Nous Could en outre observer que les cellules avec un niveau d&#39;activité similaire Hog1 pourraient afficher de façon saisissante différents résultats d&#39;expression. La combinaison de ces deux techniques nous a permis de montrer comment le remodelage lent des gènes de réponse au stress influencé le résultat de l&#39;expression au niveau d&#39;une seule cellule 7. Dans cet article, nous utilisons l&#39;expression de la p-qV STL1 journaliste induite par le choc hyper-osmotique, par exemple, de la quantification de l&#39;expression des protéines par microscopie et cytométrie de flux. La même souche soumis à NaCl 0,2 M de stress a été étudiée avec les deux techniques. Cela nous permettra de mettre en évidence certaines différences clés dans ces deux techniques très complémentaires.

<table border="1">
		<tbody>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Reagent</td>
		 </tr>
		 <tr>
			 <td>Yeast Nitrogen Base</td>
			 <td>ForMedium</td>
			 <td>CYN6202</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>CSM amino-acid mix</td>
			 <td>ForMedium</td>
			 <td>DCS0011</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Concanavalin A</td>
			 <td>GE Healthcare</td>
			 <td>17-0450-01</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Cycloheximide</td>
			 <td>Fluka Aldrich Sigma</td>
			 <td>C7698</td>
			 <td>Toxic</td>
		 </tr>
		 <tr>
			 <td>ySP9</td>
			 <td></td>
			 <td></td>
			 <td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td>
		 </tr>
		 <tr>
			 <td>pSP34</td>
			 <td></td>
			 <td></td>
			 <td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td>
		 </tr>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Material</td>
		 </tr>
		 <tr>
			 <td>Microscope</td>
			 <td>Nikon</td>
			 <td>Ti-Eclipse</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Microscope control software</td>
			 <td>Micro-manager</td>
			 <td>Ver 1.4.11</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Incubation chamber</td>
			 <td>LIS</td>
			 <td>The Box</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Fluorescence light source</td>
			 <td>Lumencor</td>
			 <td>SpectraX</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Camera</td>
			 <td>Hamamatsu</td>
			 <td>Flash 4.0</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Flow cytometer</td>
			 <td>BD</td>
			 <td>FACS calibur</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Sonicator bath</td>
			 <td>Telesonic</td>
			 <td>TUC-150</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>8-well-slide </td>
			 <td>Thermo Lab-tek</td>
			 <td>155409</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>96-well-plate</td>
			 <td>Matrical bioscience</td>
			 <td>MGB096-1-2-LG</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>FACS tube</td>
			 <td>BD Falcon</td>
			 <td>352054</td>
			 <td></td>
		 </tr>
		</tbody>
 </table>

temporal quantification of mapk induced expression in single yeast cells

La quantification de l&#39;expression des gènes au niveau d&#39;une seule cellule découvre les mécanismes de régulation nouveaux occultés dans les mesures effectuées au niveau de la population. Deux méthodes basées sur la microscopie et cytométrie en flux sont présentés pour démontrer comment ces données peuvent être acquises. L&#39;expression d&#39;un rapporteur fluorescent induit lors de l&#39;activation de la voie MAPK glycérol haute osmolarité dans la levure est utilisé comme un exemple. Les avantages spécifiques de chacune de ces méthodes sont mises en évidence. Cytométrie de flux mesure un grand nombre de cellules (10 000) et fournit une mesure directe de la dynamique de l&#39;expression protéique indépendante de la cinétique de maturation lente de la protéine fluorescente. Imagerie des cellules vivantes par microscopie est en revanche limitée à la mesure de la forme mature de la journaliste dans moins de cellules. Cependant, les ensembles de données générées par cette technique peuvent être extrêmement riches grâce à la combinaison de plusieurs journalistes et à l&#39;information spatiale et temporelleobtenu à partir de cellules individuelles. La combinaison de ces deux méthodes de mesure peut offrir de nouvelles perspectives sur la régulation de l&#39;expression de la protéine par des voies de signalisation.

Microscopie Les cellules de levure portant l&#39;expression reporter p STL1-qV (ySP9 7) ont été fixées au fond de la diapositive et bien placés sous le microscope. Les cellules ont été stimulées par l&#39;addition de milieu de contrainte directement dans le puits au cours de la session d&#39;imagerie. Cela nous permet d&#39;acquérir quelques images des cellules avant l&#39;induction voie et suivons leur sort après la stimulation. Dans le cas présent, les cellules o...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Deux méthodes complémentaires basées sur la cytométrie en flux et la microscopie sont présentées qui permettent la quantification, au niveau de la cellule unique, de la dynamique de l&#39;expression génique induits par l&#39;activation d&#39;une voie de MAPK dans la levure.

Quantification temporelle d&#39;expression MAPK induite en simples cellules de levure

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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8.4K vues.  University of Lausanne.  Deux méthodes complémentaires basées sur la cytométrie en flux et la microscopie sont présentées qui permettent la quantification, au niveau de la cellule unique, de la dynamique de l'expression génique induits par l'activation d'une voie de MAPK dans la levure. 

Vidéo: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

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

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

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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

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

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

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Expression de protéines recombinantes dans le levure méthylotrophe<emPichia pastoris></em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification des phénotypes inhibition de la croissance induite par l&#39;expression de bactéries de type III Effectors dans la levure

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

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 ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imagerie réorganisation spatiale d&#39;une voie de signalisation MAPK Utilisation du système d&#39;expression transitoire du tabac

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

L&#39;utilisation des dispositifs microfluidiques pour mesurer et cellulaire Lifespan dans Phenotypes simples cellules de levure de bourgeonnement

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...