This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. Producción del plásmido de expresión pHLsec-Gzm <ol><li> Preparar el ARN total de las células NK humanas (células primarias preparadas como en el 26 o la línea de células NK NK-92 que expresa los cinco Gzms humanos) utilizando un método de aislamiento de ARN adecuado y transcribir inversa utilizando un kit de primera cadena sintetizar ADNc, siguiendo el manufacturer` recomendaciones s. Amplificar ADNc Gzm usando PCR y el clon en pHLsec como se describe en 23 (especie de regalo de Radu Aricescu e Yvonne Jones, de la Universidad de Oxford, Reino Unido), utilizando los sitios AgeI y KpnI (Figura 1). NOTA: Utilice los siguientes cebadores indicados en la Tabla 1. </li><li> Confirmar inserciones correctas por secuenciación. Expandir los plásmidos de expresión en células DH5a y purificar utilizando un kit de aislamiento de plásmido libre de endotoxinas y siga las instrucciones del fabricante. </li><li> Resuspender los plásmidos purificados en libre de endotoxina, agua estéril a una concentración de 2 mg / ml y se almacena a -20 ° C until uso. </li></ol> 2. Expresión de Gzms en células HEK293T <ol><li> Preparación de los reactivos <ol><li> Preparar medio de cultivo estándar. Para Medio Eagle Modificado de Dulbecco (DMEM) que contenía alta glucosa (25 mM), glutamax (4 mM), piruvato de sodio (1 mM) añadir 10% de suero estándar de ternera fetal (FCS), penicilina (100 unidades / ml), estreptomicina ( 100 g / ml). </li><li> Preparar medio de transfección. Para medio de cultivo sin penicilina-estreptomicina añadir 25 g / ml de cloroquina (añadir recién en el día de la transfección de las existencias 1,000x en PBS, almacenado a -20 ° C). </li><li> Preparar medio de cultivo libre de suero: Para medio libre de suero para células HEK293 añadir glutamina (4 mM), penicilina (100 unidades / ml), estreptomicina (100 g / ml), y 2,5 mg / L anfotericina B. </li><li> Preparar las soluciones descritas en la Tabla 2 y el filtro con un filtro de 0,45 micras antes de su uso: </li></ol></li><li> Ampliando las células HEK293T <ol><li> Crecer células HEK293T en 20 ml de cultomedio ure utilizando 150 x 25 mm placas de cultivo de tejidos. </li><li> Dividir celdas a 80% de confluencia (split-relación de 1: 4, por lo general cada día 3º). Las células se adhieren libremente, que pueden separarse mecánicamente sin tripsinización por rigurosamente pipeteando arriba y abajo. </li><li> Células de la placa la noche antes de la transfección para dar 60-70% de confluencia en el día de la transfección (densidad de siembra ~ 2 x 10 5 células / ml). Un tamaño habitual preparación consiste en 20 a 25 placas de cultivo. </li></ol></li><li> Transfección de fosfato de calcio <ol><li> 1 hr antes de la transfección, cambiar el medio de cultivo estándar a 20 ml de medio de transfección. Cerca del final de esta etapa de incubación 1 h, la mezcla 400 g de ADN plásmido con 10,95 ml ddH 2 0 y 1,55 ml de 2M CaCl 2 en tubos de 50 ml (1 tubo / 5 platos). </li><li> 1 a 2 min antes de la transfección, añadir 12,5 ml de 2x HBS a 12,5 ml de la solución de ADN-calcio, mezclar por inversión de los tubos y se incuba durante 30 segundos a RT. </li><li> Añadir ªmezcla e prepara en 2.3.2 directamente a las células (5 ml por placa), gota a gota en el medio. Espolvorear uniformemente sobre toda el área, convirtiendo el medio a un color ligeramente anaranjado. </li><li> Incubar las placas de cultivo de 7 a 11 horas en una incubadora de cultivo de tejidos (37 ° C, 5% de CO2 en aire humidificado). Después de la incubación, un precipitado fino es visible. Retire el medio de transfección, enjuagar cuidadosamente con PBS precalentado y añadir 20 ml de medio de cultivo libre de suero preparado en 2.1.3. Incubar durante 72 horas en una incubadora de cultivo de tejidos. </li><li> Analizar la eficacia de transfección y expresión mediante la ejecución de una muestra (~ 20 l) del sobrenadante celular en SDS-PAGE y tinción con azul brillante de Coomassie para visualizar una banda de granzima. </li></ol></li><li> Purificación de Gzms del sobrenadante del cultivo por Nickel-IMAC <ol><li> Después de la incubación, decantar los sobrenadantes de cultivo de células en tubos de 250 ml y clara por centrifugación. Llevar a cabo una primera vuelta (400 xg, 10 min, 4 °C) que despejará el medio de células separadas. Transferir el sobrenadante en fresco 250 ml tubos y centrifugado a 4000 xg, 30 min a 4 ° C para eliminar cualquier residuo de células restante. </li><li> Añadir 5 ml de NaCl 5 M, 6,25 ml de 2 M Tris-Base (pH 8) y 1 ml de 0,25 M NiSO4 por 250 ml de sobrenadante clarificado. Filtrar el sobrenadante usando una unidad de filtro de vacío 500 ml (0,45 mm). </li><li> Equilibrar una columna de níquel-IMAC con Su-tampón de unión A (10 volúmenes de columna, CV) utilizando un sistema de FPLC adecuado. </li><li> Aplicar el sobrenadante aclarado a la columna equilibrada a un caudal de 0,5 ml / min a 4 ° C. </li><li> Después se aplicó el sobrenadante, lavar la columna con Su-tampón de unión hasta la línea base de absorbancia UV se alcanza (por lo general 10 CV). </li><li> Eluir Gzms con un 20 ml-gradiente lineal (0 a 250 mM imidazol) a una velocidad de flujo de 0,5 ml / min mientras que la recogida de 2 ml fracciones. Analizar las fracciones de elución por SDS-PAGE y tinción de Coomassie. </li></ol></li><li> Enteroquinasa (EK)el tratamiento y la limpieza final por cromatografía de intercambio catiónico <ol><li> Piscina Gzm contiene fracciones en un tubo de diálisis con ≤10 MWCO. Guarde una pequeña muestra a -20 ° C como control pre-EK. </li><li> Añadir EK en 0,02 unidades / 50 ml de sobrenadante inicial directamente a la fracción reunida en el tubo de diálisis y diálisis O / N (al menos 16 horas) a temperatura ambiente en EK-buffer (4 L, el cambio búfer una vez). </li><li> El día siguiente, analizar EK tratada Gzms en comparación con el control pre-EK por SDS-PAGE y tinción de Coomassie. </li><li> Cuando el procesamiento N-terminal es completa, cambiar tampón de diálisis a S tampón A (4 L) y se dializa durante otras 4 horas a RT. Dializado filtro (0,45 m). </li><li> Equilibrar un S-columna con tampón S A. Cargar la muestra en la columna con un caudal de 0,5 ml / min a 4 ° C. </li><li> Después de cargar la muestra, lavar la columna con tampón A hasta S UV absorbancia se alcanza la línea de base (sobre 10 CV). Eluir los Gzms con un gradiente lineal de 30 ml (150 a 1000 mM de NaCl). GzmA correolaúdes en ~ 650 mM NaCl, GzmB en ~ 700 mM NaCl y GzmM en ~ 750 mM NaCl. </li><li> Analizar las fracciones de elución por SDS-PAGE y ensayos colorimétricos (véase más adelante). Fracciones que contienen Gzms piscina y concentrado (aproximadamente 30 veces, a una concentración de aproximadamente 100 mM) en filtros de giro (15 ml, 10 MWCO). Alícuota los preparativos GZM concentrados y almacenar a -80 ° C. </li></ol></li></ol> Actividad 3. Prueba Gzm <ol><li> Preparación de los reactivos <ol><li> Preparar tampón de ensayo colorimétrico: 50 mM Tris-Base, pH 7. <ol><li> Para la medición de GzmA, añadir Na-CBZ-L-lisina tiobencilo éster (S-Bzl) (BLT) para el tampón de ensayo a una concentración final de 0,2 mM y 5,5&#39;-ditio-bis (ácido 2-nitrobenzoico) (DTNB) a una concentración de 0,22 mM. Para GzmB, incluir 0,2 mM Boc-Ala-Ala-Asp-S-Bzl mM DTNB (AAD) y 0.22. Para GzmM, incluir 0,2 mM Suc-Ala-Ala-Pro-Leu-p-nitroanilida (pNA) (AAPL). Los péptidos sintéticos se añaden de soluciones de reserva adecuadas (en DMSO, stored a -20 ° C) a la memoria intermedia recién antes del ensayo. </li></ol></li><li> Preparar tampón de ensayo de escisión: NaCl 100 mM, 50 mM Tris-Base, pH 7,5 </li></ol></li><li> Ensayos colorimétricos <ol><li> Distribuir muestras pequeñas gzm (&lt;5 l) de fracciones de elución o poblaciones concentradas en las placas de 96 pocillos de fondo plano. Incluya un control positivo (preparaciones GZM probados o crudo lisados ​​de células NK) y un control negativo (tampón solamente). </li><li> Añadir 100 l de tampón de ensayo a cada pocillo (usando una pipeta multicanal). Incubar durante 10 minutos a 37 ° C. Medir la DO a 405 nm en un lector de microplacas. </li></ol></li><li> Ensayo de escisión <ol><li> Para los ensayos de escisión utilizando sustratos purificados, co-incubar un sustrato de proteína (nativo o recombinante, 100-400 nM; algunos ejemplos de sustratos gzm eficientes se presentan en la sección de resultados y en la nota al final de esta sección) con diluciones en serie de un Gzm (a partir de 400 nM) en tampón de ensayo durante diversos tiempos.Analizar mediante SDS-PAGE y tinción con plata o Coomassie; o mediante transferencia Western usando anticuerpos específicos. </li><li> Para los ensayos de escisión utilizando lisados ​​de células, suspender 10 6 células HeLa (o cualquier línea de células tumorales disponible) en 1 ml de tampón de ensayo y se lisan por congelación en un / baño de hielo seco de etanol y descongelación a 37 ° C tres veces. Eliminar los restos celulares por centrifugación (15.000 xg durante 10 min a 4 ° C). </li><li> Co-incubar el lisado aclarado con Gzms en diferentes concentraciones y tiempos que el anterior. La escisión se detecta por inmunotransferencia utilizando anticuerpos apropiados. NOTA: Algunos ejemplos de sustratos de buena fe para que los anticuerpos comerciales están disponibles: Oferta (BH3-interacción agonista muerte de dominio) y la caspasa 3 escindida por GzmB 27,28; múltiples ribonucleoproteínas nucleares heterogéneas (hnRNPA1, A2 y U) escindidas por GzmA 29; citomegalovirus fosfoproteína 71 por GzmM 30. </li></ol></li></ol>

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P., 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=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., 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=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-[3H]acetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity.">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li></ol>

The authors have nothing to disclose.

El papel clásico y ampliamente estudiado de GzmA y GzmB es la inducción de apoptosis en células de mamífero después de su entrega citosólica por la proteína formadora de poros PFN 1. Recientemente, el espectro citotóxica de los Gzms se amplió de manera significativa a partir de células de mamíferos a las bacterias 9,10, así como a ciertos parásitos 11. Además, las funciones no clásicos, extracelulares de GzmA y GzmB así como la importancia biológica de los diversos Gzms ...

Los Gzms son una familia de serina proteasas altamente homólogas localizadas en los lisosomas especializados de CTL y células NK 1. Los gránulos citotóxicos de estas células asesinas también contienen las proteínas de la membrana de alteración PFN y GNLY que se liberan simultáneamente con los Gzms en el reconocimiento de una célula diana destinada para la eliminación de 2,3. Hay cinco Gzms en humanos (GzmA, B, H, K y M), y 10 Gzms en ratones (GzmA - G, K, M y N). GzmA y GzmB son los más abundantes y ampliamente estudiado en seres humanos y ratones 1. Sin embargo, estudios más recientes han comenzado a investigar las vías de muerte celular, así como los efectos biológicos adicionales mediadas por los otros llamados Gzms, huérfanos en la salud y la enfermedad 4. La función mejor conocida de las Gzms, en particular de GzmA y GzmB, es la inducción de la muerte celular programada en células de mamíferos cuando se entrega en las células diana por PFN 5,6. Sin embargo, Estudios más recientes también demostraron efectos extracelulares de los Gzms con profundo impacto en la regulación inmune y la inflamación, independientemente de la entrega citosólica por PFN 7,8. El espectro de las células que mueren de manera eficiente después de la entrada citosólica de las Gzms también fue recientemente se amplió a partir de células de mamíferos a las bacterias 9,10 e incluso ciertos parásitos 11. Estos recientes descubrimientos abrieron un campo completamente nuevo para los investigadores GZM. Por lo tanto, un sistema de expresión de mamíferos de alto rendimiento rentable aliviará significativamente el camino para los estudios futuros. Humanos, de ratón y de rata Gzms nativos se han purificado con éxito de la fracción de gránulos de CTL y NK células líneas 12-14. Sin embargo, en nuestras manos el rendimiento de tales técnicas de purificación está en el intervalo de menos de 0,1 mg / L de cultivo celular (observación no publicada y 12). Además, la resolución cromatográfica de un solo Gzm sin contaminación por other Gzms y / o proteínas que también están presentes en los gránulos es un reto (datos no publicados y 12,14). Gzms recombinantes se producen en bacterias, levaduras 15 16, células de insecto y en 17 incluso en las células de mamífero tales como HEK 293 18,19. Sólo los sistemas de expresión de mamífero tienen el potencial de producir enzimas recombinantes con modificaciones postraduccionales idéntica a la proteína citotóxica nativo. Modificaciones postraduccionales han sido implicados con la captación específica por endocitosis y la localización intracelular de la proteasa dentro de las células diana 20-22. Por lo tanto, mediante el uso de pHLsec 23 (una especie de regalo de Radu Aricescu e Yvonne Jones, de la Universidad de Oxford, Reino Unido) como la columna vertebral plásmido para la expresión Gzm, establecimos un sistema simple, tiempo y costo eficiente para la producción de proteínas de alto rendimiento en células HEK293T. pHLsec combina un promotor de CMV con un promotor-Β-actina de pollo; En conjunto, estos elementos demostrated el más fuerte promotor de la actividad en diversas líneas celulares 24. Además, el plásmido contiene un intrón de conejo Β-globina, señales de secreción y Kozak optimizada, una Lys-6xHis-tag y un poli-A de la señal. Los insertos se pueden clonar convenientemente entre la señal de secreción y la 6xHis-Lys-tag (Figura 1) asegurar la expresión óptima y eficacia de secreción para proteínas que carecen de dominios N-terminales apropiados. Para la expresión de los Gzms, que sustituye la secuencia señal de secreción endógena con la señal de secreción proporcionada por el vector seguido por una enteroquinasa (EK) sitio (DDDDK), de modo que el tratamiento EK activado el Gzms secretadas (Gzms activos comenzar con el N-terminal secuencia de aminoácidos IIGG 25). Adicionalmente a favor de este método, las células HEK293T crecen rápidamente en un medio de bajo precio, como medio de Eagle modificado por Dulbecco (DMEM), y son muy adecuadas para el método de transfección de fosfato de calcio rentable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

En la siguiente sección vamos a presentar una documentación completa de un preparado GzmA para iluminar el método. También nos purificamos éxito GzmB y GzmM, produciendo resultados similares en cuanto a la eficiencia y la actividad de la purificación. Sin embargo, a partir de las preparaciones posteriores sólo mostraremos algunas piezas seleccionadas de datos. Preparaciones GzmB a partir de células 293T siguiendo el protocolo actual se utilizaron en varios estudios publicados, destacando su actividad en diversos...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

Un alto rendimiento y sistema de expresión de costo-eficiente de granzimas humanos en células de mamífero

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

a-high-yield-cost-efficient-expression-system-human-granzymes

Research

JoVE Journal

Biology

9.7K vistas.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

Video: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

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 ...

Investigación

Biología

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 ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Inmunoprecipitación de cromatina (CHIP) para el ensayo dinámico de modificación de las histonas en la expresión génica activado en las células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absoluta Quantum medición del rendimiento de las muestras de polvo

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análisis de la posición del ciclo celular en células de mamífero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Análisis de Expresión de mamíferos enlazador histonas subtipos

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

La visualización de los ARNm retículo endoplásmico localizados en células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...