Gli autori ringraziano Matthias Peter e il suo gruppo presso l&#39;Istituto di Biochimica presso l&#39;ETH di Zurigo, dove sono stati sviluppati questi metodi. Questo lavoro è stato sostenuto dal National Science Foundation svizzero.

1. Misure Microscopia <ol><li> Inoculare 5 ml di terreno sintetico con il lievito. </li><li> Crescere le cellule a 30 ° C per una notte. </li><li> Misurare il diametro esterno 600 della cultura durante la notte la mattina successiva. </li><li> Diluire cultura durante la notte in mezzo sintetico 5 ml di OD 600 0.05. </li><li> Far crescere la cultura diluito a 30 ° C per almeno 4 ore. </li><li> Preparare 3 volte concentrato (0,6 M) soluzione di NaCl in terreno sintetico. </li><li> Preparare una soluzione 1 mg / ml di Concanavalina A (ConA) in PBS. </li><li> Filtrato ~ 200 ml di soluzione di ConA nella diapositiva bene. </li><li> Lasciate riposare per 30 min. </li><li> Rimuovere la soluzione ConA. </li><li> Aggiungere 150 microlitri H 2 O al pozzo. </li><li> Rimuovere H 2 O. </li><li> Misurare OD 600 di coltura cellulare. </li><li> Preparare 300 ml di coltura cellulare a OD 600 = 0,02 in una provetta da 1,5 ml. </li><li> Sonicare le cellule in un bagno di acqua per 45 sec. </li><li> Vortex brevemente la microtube. </li><li> Sonicare le cellule in un bagno di acqua per 45 sec. </li><li> Vortex brevemente la provetta. </li><li> Aggiungere 200 ml di coltura cellulare al pozzo. </li><li> Attendere 30 minuti per consentire le cellule si depositano sul fondo del pozzo. </li><li> Posizionare il vetrino bene sul microscopio. </li><li> Selezionare le impostazioni di illuminazione per il canale fluorescente da registrare. </li><li> Selezionare le impostazioni di illuminazione per due immagini in campo chiaro (uno leggermente fuori fuoco: 3 micron sotto il piano focale per permettere una corretta segmentazione delle cellule). </li><li> Selezionare gli intervalli di tempo per la misurazione time-lapse </li><li> Selezionare i campi di vista di immagine. </li><li> Avviare l&#39;acquisizione di alcuni fotogrammi. </li><li> Pausa l&#39;acquisizione di aggiungere 100 ml di 0,6 M NaCl medie. </li><li> Riprendere immagini. </li></ol> 2. Misure Citometria a flusso <ol><li> Inoculare 5 ml di terreno sintetico con il lievito. </li><li> Coltivare a 30 ° C durante la notte. </li><li> Misural&#39;OD 600 della cultura durante la notte la mattina successiva. </li><li> Diluire cultura durante la notte in mezzo sintetico 5 ml di OD 600 0.1. </li><li> Coltivare a 30 ° C per almeno 4 ore per raggiungere un OD 600 di 0,2-0,4. </li><li> Preparare la soluzione cycloheximide 1 mg / ml in H 2 O. </li><li> Preparare 3 volte concentrato (0,6 M) soluzione di NaCl in terreno sintetico. </li><li> Preparare una provetta da 1,5 ml con 100 ml di 0,6 M NaCl medio per ciascun punto di tempo da misurare. </li><li> Al tempo 0, aggiungere 200 ml di coltura cellulare per ogni provetta. </li><li> Incubare con agitazione a 30 ° C. </li><li> Al tempo T, aggiungere 30 microlitri cycloheximide a una provetta. </li><li> Ripetere passo 2.11 per tutti i punti di tempo desiderati. </li><li> Incubare tutte microtubi per almeno 45 minuti e fino a 3 ore a 30 ° C con agitazione. </li><li> Preparare tubi FACS con 400 microlitri di PBS. </li><li> Sonicare le cellule in bagnomaria per 1 min. </li><li> Vortex brevemente i microtubi. </li><li> Figlioicate le cellule in bagnomaria per 1 min. </li><li> Vortex brevemente i microtubi. </li><li> Aggiungere 100 microlitri di coltura cellulare da ciascuna provetta al corrispondente tubo FACS. </li><li> Selezionare il laser di eccitazione 488 nm e 530/30 filtro passa-banda nm per il rilevamento. </li><li> Test di non esprimere e di esprimere appieno esempio per verificare se rientrano nel campo di sensibilità del rivelatore. </li><li> Misurare 10.000 cellule per ogni campione acquisito. </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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Microscopia Il trattamento del pozzetto con ConA è un passo essenziale per assicurare una corretta rappresentazione delle cellule. Poiché ConA ha una bassa solubilità in PBS (5 mg / ml), il processo di filtrazione permette la rimozione di grandi aggregati che sono presenti nella soluzione filtrata e interferiscono con l&#39;imaging. Cellule attribuiscono relativamente fortemente alla superficie trattata e l&#39;aggiunta della soluzione indurre non dovrebbero disturbare la localizzazione dell...

Segnalazione via cascate di trasduzione spesso culmina nella espressione di proteine. La caratterizzazione di questo profilo di espressione è un elemento chiave per comprendere la funzione di percorsi biologici. L&#39;identificazione dello spettro di up-regolata proteine ​​e le dinamiche della loro attivazione può essere ottenuto attraverso diverse tecniche come micro-array, Northern blot o western blot 1-3. Tuttavia, queste tecniche media la risposta di un&#39;intera popolazione di cellule. Per comprendere la regolazione fine della espressione delle proteine, è desiderabile raccogliere misurazioni a livello di singola cellula. Idealmente, queste misure dovrebbero anche fornire dati quantitativi suscettibili di sviluppare modelli matematici della via sottostante. Microscopia e citometria a flusso sono due tecniche, che sono ideali per fornire tali misurazioni quantitative singola cellula. La codifica endogena di proteine ​​con una proteina fluorescente può be utilizzato per quantificare il loro livello di espressione 4. Tuttavia, dato che l&#39;aggiunta di una grande porzione fluorescente al terminale della proteina può renderlo non funzionale, è spesso più desiderabile generare specifici reporter di espressione basati su un promotore guida l&#39;espressione di un costrutto fluorescente. Questa proteina giornalista è esogeno al sistema cellulare e quindi non influenza gli eventi di segnalazione che stanno avvenendo nella cella. Sia microscopia e citometria a flusso sono stati ampiamente utilizzati in campo segnalamento lievito. Come esempi; collaboratori Colman-Lerner e correlati, in singole cellule, l&#39;espressione di accoppiamento reporter specifici e costitutivamente espresse mediante microscopia a quantificare il rumore nel lievito accoppiamento trasduzione del segnale cascata 5, Acar e colleghi flusso citometria utilizzati per studiare la rete di regolamentazione controllo dell&#39;espressione dei geni GAL 6. In uno studio precedente 7, abbiamo utilizzato una combinatisu queste due tecniche per studiare l&#39;espressione uscita dal glicerolo alta osmolarità (HOG) percorso in erba lievito. Questo mitogen proteina chinasi attivata (MAPK) è innescato da stress iper-osmotico. Esso determina l&#39;attivazione dei Hog1 MAPK, che trasloca nel nucleo della cellula per indurre un programma trascrizionale conseguente espressione di circa 300 geni. Per studiare questo processo, abbiamo costruito un reporter espressione basato sul promotore STL1 (un gene indotto specificamente in risposta all&#39;attività Hog1 8) che guida l&#39;espressione di una proteina fluorescente Venus quadrupla (p STL1-qv). Citofluorimetria misurazioni scoperto la presenza di due popolazioni di cellule a livello di stress intermedio (0.1 M NaCl) con solo una frazione della popolazione che esprime il reporter fluorescente. Abbiamo usato la microscopia di approfondire questo comportamento e abbiamo scoperto che questo rumore di espressione della proteina è stata governata da fattori intrinseci 9. Noi could osservano inoltre che le cellule con un analogo livello di attività Hog1 possono visualizzare profondamente diversi esiti di espressione. La combinazione di queste due tecniche ha permesso di dimostrare come la lenta ristrutturazione dei geni di risposta allo stress ha influenzato l&#39;esito espressione a livello di singola cellula 7. In questo lavoro, usiamo l&#39;espressione della p STL1-qV giornalista indotta dallo shock iper-osmotico come un esempio della quantificazione dell&#39;espressione proteica mediante microscopia e citometria a flusso. Lo stesso ceppo sottoposto a 0,2 M NaCl sforzo è stato studiato con entrambe le tecniche. Questo ci permetterà di evidenziare alcune differenze fondamentali in queste due tecniche altamente complementari.

<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 quantificazione dell&#39;espressione genica a livello di singola cellula svela nuovi meccanismi di regolamentazione oscurate nelle misurazioni effettuate a livello di popolazione. Due metodi basati sulla microscopia e citometria a flusso sono presentati per illustrare come tali dati possono essere acquisiti. L&#39;espressione di un reporter fluorescente indotto all&#39;attivazione della elevata osmolarità glicerolo MAPK in lievito è usato come esempio. I vantaggi specifici di ciascun metodo sono evidenziati. Flusso misura citometria un gran numero di cellule (10.000) e fornisce una misura diretta delle dinamiche di espressione proteica indipendente delle lenti cinetica di maturazione della proteina fluorescente. Imaging di cellule viventi mediante microscopia è invece limitato alla misura della forma maturato del reporter in meno cellule. Tuttavia, le serie di dati generati da questa tecnica possono essere estremamente ricco grazie alle combinazioni di reporter multipli e alle informazioni spaziale e temporaleottenuto da cellule singole. La combinazione di questi due metodi di misurazione in grado di fornire nuove intuizioni sulla regolazione dell&#39;espressione proteica mediante vie di segnalazione.

Microscopia Le cellule di lievito recanti l&#39;espressione giornalista STL1 p-q V (ySP9 7) sono stati attaccati al fondo del pozzo scorrevole e posti sotto il microscopio. Le cellule sono state stimolate con l&#39;aggiunta di mezzo sollecitazioni direttamente nel pozzo nel corso della sessione di imaging. Questo ci permette di acquisire alcune immagini delle cellule prima dell&#39;induzione percorso e seguiamo il loro destino dopo la stimolazione. Nella fattispecie, le cell...

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

Due metodi complementari basati sulla citometria di flusso e microscopia sono presentati che consentono la quantificazione, a livello di singola cellula, delle dinamiche di espressione genica indotte dall&#39;attivazione di una via MAPK in lievito.

La quantificazione temporale di espressione MAPK indotta in cellule di lievito singole

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 Views.  University of Lausanne.  Due metodi complementari basati sulla citometria di flusso e microscopia sono presentati che consentono la quantificazione, a livello di singola cellula, delle dinamiche di espressione genica indotte dall'attivazione di una via MAPK in lievito. 

Video: 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Part 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

Espressione di proteine ​​ricombinanti nel lievito Methylotrophic 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.

Identificazione dei fenotipi inibizione della crescita indotta da Espressione di effettori batterica di tipo III nel lievito

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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 Riorganizzazione spaziale di una via di segnalazione MAPK Utilizzando il sistema transitorio Expression Tabacco

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.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

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