Die Autoren danken Matthias Peter und seine Gruppe am Institut für Biochemie an der ETH in Zürich, wo diese Verfahren entwickelt worden. Diese Arbeit wurde durch den Schweizerischen Nationalfonds unterstützt.

1. Mikroskopie-Messungen <ol><li> Impfen 5 ml synthetisches Medium mit Hefe. </li><li> Zellen wachsen bei 30 ° C über Nacht. </li><li> Messen Sie die OD 600 der Übernachtkultur am nächsten Morgen. </li><li> Verdünnen Nacht-Kultur in 5 ml synthetisches Medium bis DA 600 0,05. </li><li> Wachsen die verdünnte Kultur bei 30 ° C für mindestens 4 Stunden. </li><li> Bereiten Sie 3-fach konzentriert (0,6 M) NaCl-Lösung in synthetischem Medium. </li><li> Vorbereitung einer 1 mg / ml Lösung von Concanavalin A (ConA) in PBS. </li><li> Filtrat ~ 200 ul ConA-Lösung in die gut Rutsche. </li><li> Lassen Sie stehen für 30 min. </li><li> ConA-Lösung entfernen. </li><li> In 150 ul H 2 O zum Brunnen. </li><li> Entfernen H 2 O </li><li> OD 600-Messung der Zellkultur. </li><li> Planen 300 ul Zellkultur bei einer OD 600 = 0,02 in einem 1,5 ml Mikroröhrchen. </li><li> Beschallen der Zellen in einem Wasserbad für 45 Sekunden. </li><li> Vortex kurz die microtube. </li><li> Beschallen der Zellen in einem Wasserbad für 45 Sekunden. </li><li> Vortex kurz den Mikroröhrchen. </li><li> Geben Sie 200 ul der Zellkultur zum Brunnen. </li><li> Warten Sie 30 min die Zellen auf dem Boden der gut absetzen zu lassen. </li><li> Legen Sie die Folie auch auf dem Mikroskop. </li><li> Wählen Sie die Beleuchtungseinstellungen für die Leuchtstoff Kanal aufgezeichnet werden. </li><li> Wählen Sie die Beleuchtungseinstellungen für zwei Hellfeld-Bilder (ein etwas unscharf: 3 um unterhalb der Fokusebene, für eine angemessene Segmentierung der Zellen zu ermöglichen). </li><li> Wählen Sie die Zeitintervalle für die Zeitraffer-Messung </li><li> Wählen Sie die Felder der Ansicht zu Bild. </li><li> Starten Sie die Aufnahme von ein paar Frames. </li><li> Pause den Erwerb, die 100 ul von 0,6 M NaCl Medium hinzufügen. </li><li> Resume-Bildgebung. </li></ol> 2. Durchflusszytometrie Messungen <ol><li> Impfen 5 ml synthetisches Medium mit Hefe. </li><li> Wachsen bei 30 ° C über Nacht. </li><li> Messendie OD 600 der Übernacht-Kultur am nächsten Morgen. </li><li> Verdünnen Nacht-Kultur in 5 ml synthetisches Medium bis DA 600 0.1. </li><li> Wachsen bei 30 ° C für mindestens 4 Stunden bis zu einer OD 600 von 0,2-0,4 zu erreichen. </li><li> Vorbereitung Cycloheximid-Lösung 1 mg / ml in H 2 O. </li><li> Bereiten Sie 3-fach konzentriert (0,6 M) NaCl-Lösung in synthetischem Medium. </li><li> Vorbereitung einer 1,5 ml-Mikroröhrchen mit 100 &amp;mgr; l 0,6 M NaCl Medium für jeden Zeitpunkt gemessen werden. </li><li> Zum Zeitpunkt 0, 200 &amp;mgr; l Zellkultur jedem Mikroröhrchen. </li><li> Inkubation mit bei 30 ° C schüttelnd </li><li> Zum Zeitpunkt T, fügen Sie 30 ul Cycloheximid zu einem Mikroröhrchen. </li><li> Wiederholen Sie Schritt 2.11 für alle gewünschten Zeitpunkten. </li><li> Alle Röhrchen Inkubation für mindestens 45 min und bis zu 3 h bei 30 ° C unter Schütteln. </li><li> Bereiten FACS-Röhrchen mit 400 ul PBS. </li><li> Beschallen die Zellen im Wasserbad für 1 min. </li><li> Vortex kurz die Mikroröhrchen. </li><li> Sohnfindliche die Zellen im Wasserbad für 1 min. </li><li> Vortex kurz die Mikroröhrchen. </li><li> 100 ul Zellkultur von jedem Mikroröhrchen in das entsprechende FACS-Röhrchen. </li><li> Wählen Sie die 488 nm Anregungslaser und 530/30 nm Bandpassfilter für die Erkennung. </li><li> Test-nicht-exprimierenden und Proben voll zum Ausdruck, um zu überprüfen, ob sie in der Detektor-Empfindlichkeitsbereich fallen. </li><li> Messen 10.000 Zellen für jede erworbene Probe. </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>

Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Mikroskopie Die Behandlung der mit ConA und ist ein wesentlicher Schritt, um eine richtige Abbildung der Zellen sicherzustellen. Da ConA eine geringe Löslichkeit in PBS (5 mg / ml), kann die Filtration der Entfernung von großen Aggregaten, die in der ungefilterten Lösung vorliegen und stören die Bildgebung. Zellen befestigen relativ stark auf die behandelte Oberfläche und die Zugabe der Lösung zu induzieren nicht die Lokalisierung der Zellen stören, was eine kontinuierliche Verfolgung vo...

Signalisierung über transduktionskaskaden oft gipfelt in der Expression von Proteinen. Die Charakterisierung des Expressionsprofils ist ein Schlüsselelement für das Verständnis der Funktion der biologischen Wege. Die Identifizierung des Spektrums hochreguliert Proteine ​​und die Dynamik ihrer Aktivierung kann durch verschiedene Techniken wie Mikroarrays, Northern-Blots und Western Blots 1-3 erreicht werden. , Im Durchschnitt jedoch diese Techniken die Antwort einer ganzen Population von Zellen. Um die Feinregulierung der Expression von Proteinen zu verstehen, ist es wünschenswert, Messungen auf Einzelzellebene zu sammeln. Idealerweise sollten diese Messungen auch quantitative Daten zugänglich mathematische Modelle des Basiswegs zu entwickeln. Mikroskopie und Durchflusszytometrie sind zwei Techniken, die ideal geeignet sind, wie quantitative Einzelzellmessungen liefern. Die endogenen Markierung von Proteinen mit einem fluoreszierenden Protein kann bE verwendet werden, um ihre Expression Level 4 zu quantifizieren. Da die Zugabe einer großen fluoreszierenden Gruppe am Terminus des Proteins kann es machen nicht-funktionellen, ist es jedoch oft wünschenswert, spezifische Expression Reporter basierend auf einem Promotor, der die Expression eines Fluoreszenz Konstrukt zu erzeugen. Dieses Reporter-Protein exogen zu dem zellularen System und daher nicht die Signalisierungsereignisse, die in der Zelle stattfinden, beeinflussen. Sowohl Mikroskopie und Durchflusszytometrie wurden weitgehend im Signalisierungsfeld Hefe verwendet. Als Beispiele; Colman-Lerner und Mitarbeiter korreliert sind, in einzelnen Zellen die Expression von spezifischen Paarung und konstitutiv exprimiert Reportern durch Mikroskopie, um den Lärm in Hefe Paarung Signalübertragungskaskade 5 quantifizieren, Acar und Kollegen verwendeten Durchflusszytometrie, um die regulatorische Netzwerk studieren Steuern der Expression der GAL-Gene 6. In einer früheren Studie 7 haben wir eine Kombina verwendetauf dieser zwei Techniken, um die Expressionsleistung aus der Hoch Osmolarität Glycerin (HOG) Weg in Hefezellen zu studieren. Diese Mitogen-aktivierte Proteinkinase (MAPK)-Weg wird durch Hyper osmotischen Stress ausgelöst. Es führt zu der Aktivierung der MAPK Hog1, die in den Kern der Zelle transloziert, ein Transkriptionsprogramm, was zur Expression von etwa 300 Gene zu induzieren. Um diesen Prozess zu untersuchen, haben wir einen Ausdruck Reporter bezogen auf das STL1 Promotor (ein Gen, das unter den Hog1 Aktivität 8 induziert), die Expression von einem Vierfach-Venus fluoreszierendes Protein (p-STL1 qV) entwickelt hatten. Durchflusszytometrie Messungen entdeckt die Anwesenheit von zwei Populationen von Zellen bei mittleren Belastungsniveau (0,1 M NaCl) nur mit einem Bruchteil der Bevölkerung die fluoreszierenden Reporter exprimieren. Wir verwendeten Mikroskopie dieses Verhalten weiter zu untersuchen und entdeckt, dass dieses Geräusch in der Proteinexpression wurde durch intrinsische Faktoren 9 geregelt. Wir could beobachten weiter, dass Zellen, die mit einem ähnlichen Niveau von Hog1 Aktivität könnte auffallend unterschiedliche Expressionsergebnisse anzuzeigen. Die Kombination dieser beiden Techniken konnten wir zeigen, wie die langsamen Umbau der Stressantwortgene beeinflusst die Expression Ergebnis auf Einzelzellebene 7. In diesem Papier verwenden wir die Expression des p-STL1 qV Reporter durch hyper-osmotischen Schock als ein Beispiel für die Quantifizierung der Proteinexpression induziert durch Mikroskopie und Durchflusszytometrie. Der gleiche Stamm bis 0,2 M NaCl beansprucht wurde mit beiden Methoden untersucht. Dies wird es uns ermöglichen, einige der wichtigsten Unterschiede in diesen beiden Techniken ergänzen sich zu markieren.

<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

Die Quantifizierung der Genexpression auf Einzelzellebene deckt neuen Regulationsmechanismen bei Messungen auf Bevölkerungsebene durchgeführt verdeckt. Zwei Methoden, die auf Mikroskopie und Durchflusszytometrie werden dargestellt, um zu demonstrieren, wie solche Daten erfasst werden. Die Expression eines fluoreszierenden Reporter bei Aktivierung der hohe Osmolarität Glycerin MAPK in Hefe induziert wird als Beispiel verwendet. Die spezifischen Vorteile der einzelnen Verfahren werden hervorgehoben. Durchflusscytometrie misst eine Vielzahl von Zellen (10.000) und liefert ein direktes Maß für die Dynamik der Protein-Expression unabhängig von der langsamen Reifung Kinetik des fluoreszierenden Proteins. Abbildung von lebenden Zellen durch Mikroskopie ist im Gegensatz zu der Messung der gereiften Form der Reporter in weniger Zellen beschränkt. Jedoch können die Datensätze durch diese Technik erzeugt extrem reich durch die Kombinationen von mehreren Reportern und um die räumliche und zeitliche Informationen seinaus Einzelzellen erhalten. Die Kombination dieser beiden Messmethoden können neue Erkenntnisse über die Regulation der Proteinexpression durch Signalwege zu liefern.

Mikroskopie Hefe-Zellen, die das Reportergen Expression STL1 p-qV (ySP9 7) zu dem Boden des Bohrlochs-Schlitten angebracht ist und unter das Mikroskop gelegt. Die Zellen wurden während des Verlaufs der Bildgebungssitzung durch Zugabe von Stress Medium direkt in das Bohrloch gefördert. Dies ermöglicht es uns, ein paar Bilder von den Zellen vor Induktion Weg erwerben und folgen Sie ihr Schicksal nach der Stimulation. Im vorliegenden Fall wurden die Zellen für ca. 2 h mit e...

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

Zwei komplementäre Verfahren basierend auf Durchflusszytometrie und Mikroskopie vorgestellt, die die Quantifizierung auf der Ebene einer einzelnen Zelle, die Dynamik der Genexpression durch die Aktivierung eines MAPK-Weg in Hefe induziert ermöglichen.

Zeitliche Quantifizierung der MAPK induzierte Expression in Einzel-Hefezellen

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

temporal-quantification-mapk-induced-expression-single-yeast

Research

JoVE Journal

Biology

8.4K Aufrufe.  University of Lausanne.  Zwei komplementäre Verfahren basierend auf Durchflusszytometrie und Mikroskopie vorgestellt, die die Quantifizierung auf der Ebene einer einzelnen Zelle, die Dynamik der Genexpression durch die Aktivierung eines MAPK-Weg in Hefe induziert ermöglichen. 

Serie: 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.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 1

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

Forschung

Biologie

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.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 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.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 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.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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 von rekombinanten Proteinen in der methylotrophen Hefe Pichia 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 of Growth Inhibition Phänotypen durch Expression von bakteriellen Typ III Effektoren in Hefe Induced

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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 der globalen RNA-Synthese auf der Einzelzellebene folgende 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.

Imaging Räumliche Reorganisation eines MAPK Signalweg Verwendung des Tabak Transient Expression System

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.

Mikrofluidik-Geräte mit Measure Lebensdauer und Cellular Phänotypen in Einzel Budding Hefezellen

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