Los autores agradecen a Matthias Peter y su grupo en el Instituto de Bioquímica de la ETH en Zürich, donde se han desarrollado estos métodos. Este trabajo ha sido financiado por la Fundación Nacional de Ciencia de Suiza.

1. Medidas de Microscopía <ol><li> Inocular 5 ml de medio sintético con levadura. </li><li> Se cultivan las células a 30 º C durante la noche. </li><li> Mida la OD 600 de la cultura de la noche a la mañana siguiente. </li><li> Diluir la cultura durante la noche en 5 ml de medio sintético a OD 600 0.05. </li><li> Mantener el cultivo diluido a 30 ° C durante al menos 4 horas. </li><li> Preparar 3 veces concentrado (0,6 M) una solución de NaCl en medio sintético. </li><li> Preparar una solución de 1 mg / ml de Concanavalina A (ConA) en PBS. </li><li> Filtrado ~ 200 l de solución de ConA en la diapositiva también. </li><li> Deje reposar durante 30 min. </li><li> Retire la solución ConA. </li><li> Añadir 150 l de H 2 O al pozo. </li><li> Retire H 2 O. </li><li> Mida OD 600 de cultivo celular. </li><li> Preparar 300 l de cultivo celular a OD 600 = 0,02 en un microtubo de 1.5 ml. </li><li> Sonicar las células en un baño de agua durante 45 segundos. </li><li> Vortex brevemente la microtube. </li><li> Sonicar las células en un baño de agua durante 45 segundos. </li><li> Vortex brevemente el microtubo. </li><li> Añadir 200 l de cultivo celular para el pozo. </li><li> Espere 30 minutos para dejar que las células se depositan en el fondo del pozo. </li><li> Situar el porta bien en el microscopio. </li><li> Seleccione los ajustes de iluminación para el canal de fluorescencia a grabar. </li><li> Seleccionar los ajustes de iluminación para dos imágenes de campo claro (uno ligeramente fuera de foco: 3 micras por debajo del plano focal para permitir una segmentación adecuada de las células). </li><li> Seleccione los intervalos de tiempo para la medición del tiempo-lapso </li><li> Seleccione los campos de visión de la imagen. </li><li> Iniciar la adquisición de una serie de imágenes. </li><li> Pausa la adquisición de añadir los 100 l de 0,6 M NaCl medio. </li><li> Reanudar la proyección de imagen. </li></ol> 2. Citometría de Flujo Medidas <ol><li> Inocular 5 ml de medio sintético con levadura. </li><li> Haga crecer a 30 º C durante la noche. </li><li> Medidael OD 600 de la cultura de la noche a la mañana siguiente. </li><li> Diluir la cultura durante la noche en 5 ml de medio sintético a OD 600 0.1. </li><li> Crece a 30 ° C durante al menos 4 horas para llegar a un OD 600 de 0,2 a 0,4. </li><li> Preparar la solución de cicloheximida 1 mg / ml en H 2 O. </li><li> Preparar 3 veces concentrado (0,6 M) una solución de NaCl en medio sintético. </li><li> Preparar un microtubo de 1,5 ml con 100 l de 0,6 M NaCl medio para cada punto de tiempo para ser medidos. </li><li> En el tiempo 0, añadir 200 l de cultivo celular a cada microtubo. </li><li> Incubar con agitación a 30 ° C. </li><li> En el momento T, añadir 30 l de cicloheximida a un microtubo. </li><li> Repita paso 2,11 para todos los puntos de tiempo deseados. </li><li> Incubar todos los microtubos durante al menos 45 min y hasta 3 horas a 30 º C con agitación. </li><li> Preparar tubos de FACS con 400 l de PBS. </li><li> Sonicar las células en baño de agua durante 1 min. </li><li> Vortex brevemente los microtubos. </li><li> Hijoicar las células en baño de agua durante 1 min. </li><li> Vortex brevemente los microtubos. </li><li> Añadir 100 l de cultivo celular de cada microtubo a su correspondiente tubo de FACS. </li><li> Seleccione el láser de excitación 488 nm y 530/30 nm de paso de banda de filtro para la detección. </li><li> Prueba de que no expresa y expresar plenamente la muestra para verificar si se caen en el rango de sensibilidad del detector. </li><li> Medir 10.000 células para cada muestra adquirida. </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>

Los autores declaran que no tienen intereses financieros en competencia.

Microscopía El tratamiento del pozo con ConA es un paso esencial para asegurar una imagen adecuada de las células. Debido a ConA tiene baja solubilidad en PBS (5 mg / ml), el proceso de filtración permite la eliminación de grandes agregados que están presentes en la solución sin filtrar y interfieren con la formación de imágenes. Las células se adhieren relativamente fuerte a la superficie tratada y la adición de la solución de la inducción no deben perturbar la localización de las...

La señalización a través de cascadas de transducción menudo culmina en la expresión de proteínas. La caracterización de este perfil de expresión es un elemento clave en la comprensión de la función de las vías biológicas. La identificación de espectro de hasta reguladas proteínas y la dinámica de su activación se puede lograr mediante diversas técnicas como la micro-arrays, transferencias Northern o transferencias Western 1-3. Sin embargo, estas técnicas promedio de la respuesta de toda una población de células. Para entender la regulación fina de la expresión de proteínas, es deseable para reunir mediciones en el nivel de células individuales. Lo ideal sería que estas medidas también deben proporcionar datos cuantitativos susceptibles de desarrollar modelos matemáticos de la vía subyacente. Microscopía y citometría de flujo son dos técnicas, que son ideales para ofrecer este tipo de mediciones cuantitativas de células individuales. El etiquetado endógena de proteínas con una proteína fluorescente puede be utilizado para cuantificar su nivel de expresión 4. Sin embargo, ya que la adición de un resto fluorescente grande en la parte terminal de la proteína puede hacer que no sea funcional, a menudo es más deseable generar reporteros de expresión específicos basados ​​en un promotor que dirige la expresión de una construcción fluorescente. Esta proteína indicadora es exógeno al sistema celular y por lo tanto no influye en los eventos de señalización que están teniendo lugar en la célula. Tanto la microscopía y citometría de flujo han sido ampliamente utilizados en el campo de la señalización de la levadura. Como ejemplos, los compañeros de trabajo-Colman Lerner y correlacionados, en celdas individuales, la expresión de apareamiento periodistas específicos y constitutivamente expresadas por microscopía de cuantificar el ruido en el apareamiento de levadura de transducción de señales en cascada 5, Acar y sus colegas utilizaron citometría de flujo para el estudio de la red de regulación el control de la expresión de los genes GAL 6. En un estudio anterior 7, hemos utilizado un combinatien una de estas dos técnicas para estudiar la salida de la expresión de la alta osmolaridad de glicerol (HOG) y la vía de levadura en ciernes. Esta proteína quinasa activada por mitógenos (MAPK) es provocada por el estrés hiper-osmótica. Es el resultado de la activación de las MAPK Hog1, que se transloca al núcleo de la célula para inducir un programa transcripcional que resulta en la expresión de aproximadamente 300 genes. Para estudiar este proceso, habíamos diseñado un reportero de expresión basado en el promotor STL1 (un gen inducido específicamente en respuesta a la actividad Hog1 8) que dirige la expresión de una proteína fluorescente Venus cuádruple (p STL1-qV). La citometría de flujo mediciones revelaron la presencia de dos poblaciones de células en el nivel de la tensión intermedia (NaCl 0,1 M) con sólo una fracción de la población que expresa el indicador fluorescente. Se utilizó microscopía de investigar más a fondo este comportamiento y descubrimos que este ruido en la expresión de proteínas se rige por factores intrínsecos 9. Nos COUld observan, además, que las células con un nivel similar de actividad Hog1 podrían mostrar resultados sorprendentemente diferentes de expresión. La combinación de estas dos técnicas nos ha permitido demostrar cómo la lenta remodelación de genes de respuesta al estrés influyó en el resultado de expresión a nivel de células individuales 7. En este trabajo, utilizamos la expresión de la p-STL1 qV reportero inducida por choque hiper-osmótica como un ejemplo de la cuantificación de la expresión de proteínas por microscopía y citometría de flujo. La misma cepa sometida a estrés 0,2 M de NaCl se estudió con ambas técnicas. Esto nos permitirá poner de relieve algunas diferencias clave en estas dos técnicas altamente complementarias.

<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 cuantificación de la expresión génica a nivel de células individuales descubre nuevos mecanismos de regulación oscurecidas en las mediciones realizadas a nivel de población. Se presentan dos métodos basados ​​en la microscopía y citometría de flujo para demostrar cómo se pueden adquirir esos datos. La expresión de un indicador fluorescente inducida tras la activación de la alta osmolaridad MAPK vía de glicerol en la levadura se utiliza como un ejemplo. Se destacan las ventajas específicas de cada método. La citometría de flujo mide un gran número de células (10.000) y proporciona una medida directa de la dinámica de la expresión de la proteína independiente de la cinética lenta maduración de la proteína fluorescente. Obtención de imágenes de células vivas mediante microscopía es por el contrario limitado a la medición de la forma madurado del reportero en un menor número de células. Sin embargo, los conjuntos de datos generados por esta técnica pueden ser extremadamente rico gracias a la combinación de varios periodistas ya la información espacial y temporalobtenido a partir de células individuales. La combinación de estos dos métodos de medición puede proporcionar nuevos conocimientos sobre la regulación de la expresión de proteínas por las vías de señalización.

Microscopía Las células de levadura que llevan el reportero de la expresión de P-STL1 qV (ySP9 7) se adjunta a la parte inferior del pozo-portaobjetos y se coloca bajo el microscopio. Las células se estimularon mediante la adición de medio de estrés directamente en el pozo durante el curso de la sesión de formación de imágenes. Esto nos permite adquirir unas pocas imágenes de las células antes de la inducción vía y seguir su destino después de la estimulación....

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

Dos métodos complementarios basados ​​en citometría de flujo y microscopía se presentan que permiten la cuantificación, a nivel de una sola célula, de la dinámica de la expresión génica inducidos por la activación de una vía MAPK en la levadura.

La cuantificación temporal de la expresión de MAPK inducida en células individuales de levadura

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 vistas.  University of Lausanne.  Dos métodos complementarios basados ​​en citometría de flujo y microscopía se presentan que permiten la cuantificación, a nivel de una sola célula, de la dinámica de la expresión génica inducidos por la activación de una vía MAPK en la levadura. 

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.

Infección por el virus vaccinia y Análisis temporal de la expresión génica 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 ...

Investigación

Biología

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.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 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.

Infección por el virus vaccinia y Análisis temporal de la expresión génica 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.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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.

Expresión de proteínas recombinantes en la levadura metilotrófica 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.

Identificación de los fenotipos de inhibición del crecimiento inducida por la expresión de tipo bacteriana efectores III en la levadura

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.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes 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.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

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.

La formación de imágenes reorganización espacial de una vía de señalización MAPK Usando el sistema de expresión transitoria del Tabaco

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.

El uso de dispositivos de microfluidos para Medir Lifespan y Celular fenotipos en células individuales levadura en ciernes

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