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. Production du plasmide d&#39;expression pHLsec-GZM <ol><li> Préparation de l&#39;ARN total à partir de cellules NK humaines (cellules primaires préparées comme dans 26 ou la lignée de cellules NK NK-92 exprimant les cinq Gzms humaines) en utilisant un procédé d&#39;isolement d&#39;ARN appropriée et transcription inverse en utilisant un premier brin kit ADNc de synthétiser la suite de la manufacturer` s recommandations. Amplifier GZM ADNc par PCR et clone dans pHLsec comme décrit dans 23 (don genre de Radu Aricescu et Yvonne Jones, Université d&#39;Oxford, Royaume-Uni) en utilisant les sites Agel et Kpnl (Figure 1). REMARQUE: Utilisez les amorces suivantes indiquées dans le tableau 1. </li><li> Confirmez inserts correctes par séquençage. Développez les plasmides d&#39;expression dans des cellules DH5a et purifier en utilisant un kit d&#39;isolement de plasmide libre endotoxine et suivez les instructions du fabricant. </li><li> Reprendre les plasmides purifiés dans de l&#39;eau stérile sans endotoxine à une concentration de 2 mg / ml et conserver à -20 ° C until utilisation. </li></ol> 2. Expression de Gzms dans des cellules HEK293T <ol><li> Préparation des réactifs <ol><li> Préparer le milieu de culture standard. Pour milieu Eagle modifié de Dulbecco (DMEM) contenant taux élevé de glucose (25 mM), Glutamax (4 mM), du pyruvate de sodium (1 mM) ajouter 10% de sérum standard de veau foetal (FCS), la pénicilline (100 unités / ml), la streptomycine ( 100 ug / ml). </li><li> Préparer milieu de transfection. Pour milieu de culture sans pénicilline-streptomycine ajouter 25 pg / ml chloroquine (ajouter fraîchement sur le jour de la transfection de 1,000x stocks dans PBS, stockés à -20 ° C). </li><li> Préparer un milieu de culture exempt de sérum: A un milieu sans sérum pour cellules HEK293 ajouter de la glutamine (4 mM), de la pénicilline (100 unités / ml), streptomycine (100 ug / ml), et 2,5 mg / L de l&#39;amphotéricine B. </li><li> Préparer les solutions décrites dans le tableau 2 et le filtre avec un filtre de 0,45 um avant utilisation: </li></ol></li><li> Expansion des cellules HEK293T <ol><li> Cultiver les cellules HEK293T dans 20 ml culteSupport d&#39;ure utilisant 150 x 25 mm boîtes de culture de tissu. </li><li> Cellules Split à 80% de confluence (split-ratio de 1: 4, généralement tous les 3 ème jour). Cellules attachent vaguement, ils peuvent être détachés mécaniquement sans traitement à la trypsine par pipetage rigoureusement haut et en bas. </li><li> cellules de la plaque avant la transfection nuit pour donner 60 à 70% de confluence au jour de la transfection (densité d&#39;ensemencement ~ 2 x 10 5 cellules / ml). Une taille de la préparation habituelle consiste de 20 à 25 boîtes de culture. </li></ol></li><li> La transfection au phosphate de calcium <ol><li> 1 h avant la transfection, remplacer le milieu de culture standard à 20 ml de milieu de transfection. Près de la fin de cette étape d&#39;incubation de 1 heure, mélanger 400 ug d&#39;ADN plasmidique avec 10,95 ml ddH 2 0 et 1,55 ml de 2M CaCl2 en tubes de 50 ml (1 tube / 5 plats). </li><li> 1 à 2 min avant la transfection, ajouter 12,5 ml 2x HBS à 12,5 ml de la solution ADN-Calcium, mélanger par inversion des tubes et incuber pendant 30 secondes à la température ambiante. </li><li> Ajouter èmee mélange préparé en 2.3.2 directement aux cellules (5 ml par boîte), goutte à goutte dans le milieu. Saupoudrer uniformément sur toute la surface, en tournant le support à une couleur légèrement orangée. </li><li> Incuber les boîtes de culture de 7 à 11 h dans un incubateur de culture de tissu (37 ° C, 5% de CO2 dans de l&#39;air humidifié). Après l&#39;incubation, une amende précipité est visible. Retirez le milieu de transfection, rincer soigneusement préchauffé PBS et ajouter 20 ml de milieu de culture sans sérum préparé à 2.1.3. Incuber pendant 72 heures dans un incubateur de culture de tissu. </li><li> Analyser l&#39;efficacité de transfection et l&#39;expression par l&#39;exécution d&#39;un échantillon (~ 20 ul) du surnageant de cellule sur SDS-PAGE et coloration avec du bleu brillant de Coomassie pour visualiser une bande de granzyme. </li></ol></li><li> Purification de Gzms de surnageant de culture par Nickel-IMAC <ol><li> Après l&#39;incubation, décanter le surnageant de culture de cellules dans 250 ml tubes et claire par centrifugation. Effectuer une première rotation (400 xg, 10 min, 4 °C) qui va effacer le milieu à partir des cellules individuelles. Transférer le surnageant dans fraîches 250 ml tubes et tournent à 4000 g, 30 min à 4 ° C pour éliminer les débris de cellules restant. </li><li> Ajouter 5 ml de NaCl 5 M, 6,25 ml de 2 M de Tris-Base (pH 8) et 1 ml de 0,25 M NiSO 4 par 250 ml de surnageant clarifié. Filtrer le surnageant à l&#39;aide d&#39;une unité de filtration sous vide de 500 ml (0,45 mm). </li><li> Equilibrer une colonne nickel-IMAC avec Sa liaison tampon A (10 volumes de colonne, CV) en utilisant un système FPLC approprié. </li><li> Appliquer le surnageant clarifié à la colonne équilibrée à un débit de 0,5 ml / min à 4 ° C. </li><li> Après le surnageant a été appliqué, colonne de lavage avec Sa liaison tampon jusqu&#39;à ce que l&#39;absorbance UV de référence est atteint (généralement 10 CV). </li><li> Gzms éluées avec un gradient de 20 ml-linéaire (0 à 250 mM imidazole) à un débit de 0,5 ml / min tout en recueillant des fractions de 2 ml. Analyser les fractions d&#39;élution par SDS-PAGE et coloration au bleu de Coomassie. </li></ol></li><li> Enterokinase (EK)le traitement et le nettoyage final par Chromatographie d&#39;échange de cations <ol><li> Piscine GZM fractions contenant dans un tube de dialyse MWCO avec ≤10. Stocker un petit échantillon à -20 ° C que le contrôle pré-EK. </li><li> Ajouter EK à 0,02 unité / ml de 50 surnageant initial directement à la fraction groupée dans le tube de dialyse et dialyse O / N (au moins 16 heures) à la température ambiante dans EK-tampon (4 L, le changement tampon une fois). </li><li> Le lendemain, analyser EK Gzms traitée par rapport à la pré-EK contrôle par SDS-PAGE et coloration au bleu de Coomassie. </li><li> Lorsque le traitement N-terminal est terminée, modifier un tampon de dialyse à tampon A S (4 L) et on dialyse pendant 4 heures à température ambiante. Filtre dialysat (0,45 um). </li><li> Équilibrer une colonne S-S avec du tampon de l&#39;échantillon A. charger sur la colonne avec à un débit de 0,5 ml / min à 4 ° C. </li><li> Après chargement de l&#39;échantillon, laver la colonne avec le tampon de S A jusqu&#39;à ce que la ligne de base UV absorbance est atteint (environ 10 CV). Éluer les Gzms avec un gradient linéaire de 30 ml (150 à 1 000 mM de NaCl). GZMA eluths à ~ 650 mM de NaCl, GZMB à ~ 700 mM de NaCl et GzmM à ~ 750 mM de NaCl. </li><li> Analyser élution fractions par SDS-PAGE et essais colorimétriques (voir ci-dessous). Piscine fractions contenant Gzms et concentré (environ 30 fois, à une concentration d&#39;environ 100 pM) dans les filtres de rotation (15 ml, 10 MWCO). Aliquoter les préparatifs GZM concentrés et conserver à -80 ° C. </li></ol></li></ol> 3. Test de l&#39;activité GZM <ol><li> Préparation des réactifs <ol><li> Préparer le tampon de dosage colorimétrique: 50 mM de Tris-Base, pH 7. <ol><li> Pour la mesure de GZMA, ajouter Na-CBZ-L-Lysine thiobenzyle ester (S-Bzl) (BLT) pour le tampon d&#39;essai à une concentration finale de 0,2 mM et 5,5&#39;-dithio-bis (acide 2-nitrobenzoïque) (DTNB) à une concentration de 0,22 mM. Pour GZMB, inclure 0,2 mM Boc-Ala-Ala-Asp-S-Bzl (AAD) et 0,22 mM DTNB. Pour GzmM, y compris 0,2 mM de Suc-Ala-Ala-Pro-Leu-p-nitroanilide (pNA) (AAPL). Les peptides synthétiques sont ajoutés à partir de solutions d&#39;achat d&#39;actions appropriées (dans le DMSO, stored à -20 ° C) dans la mémoire tampon fraîchement avant l&#39;essai. </li></ol></li><li> Préparer le tampon de dosage de clivage: 100 mM de NaCl, 50 mM de Tris-Base, pH 7,5 </li></ol></li><li> Dosages colorimétriques <ol><li> Distribuez de petits échantillons GZM (&lt;5 pi) de fractions d&#39;élution ou des stocks concentrés dans les plaques à 96 puits à fond plat. Inclure un contrôle positif (GZM préparations testées ou brut lysats de cellules NK) et un contrôle négatif (tampon seulement). </li><li> Ajouter 100 ul de tampon de dosage à chaque puits (à l&#39;aide d&#39;une pipette multi-canaux). Incuber pendant 10 min à 37 ° C. Mesurer la DO à 405 nm dans un lecteur de microplaques. </li></ol></li><li> Dosage Décolleté <ol><li> Pour les essais de clivage à l&#39;aide de substrats purifiés, co-incuber un substrat protéique (native ou recombinante, de 100 à 400 nM; quelques exemples de substrats de GZM efficaces sont présentés dans la section de résultat et à la note à la fin de cette section) avec des dilutions en série d&#39;un GZM (à partir de 400 nM) dans du tampon analytique pour différents moments.Analyse par SDS-PAGE et coloration à l&#39;argent ou au bleu de Coomassie; ou par Western blot en utilisant des anticorps spécifiques. </li><li> Pour les essais de clivage à l&#39;aide de lysats de cellules, de suspendre 10 6 cellules HeLa (ou toute lignée cellulaire de tumeur disponible) dans 1 ml de tampon de dosage et lyse par congélation dans un / bain de glace sèche de l&#39;éthanol et décongélation à 37 ° C trois fois. Éliminer les débris cellulaires par centrifugation (15 000 xg pendant 10 min à 4 ° C). </li><li> Co-incuber le lysat clarifié avec Gzms à diverses concentrations et heures ci-dessus. Le clivage est détecté par immunotransfert en utilisant des anticorps appropriés. NOTE: Quelques exemples de substrats de bonne foi pour laquelle les anticorps commerciaux sont disponibles: Offre (BH3-interaction agoniste domaine de mort) et de la caspase 3 clivée par GZMB 27,28; plusieurs ribonucléoprotéines nucléaires hétérogènes (hnRNPA1, A2 et U) clivées par GZMA 29; cytomégalovirus phosphoprotéine 71 par 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.

Le rôle classique et d&#39;études approfondies et de GZMA GZMB est l&#39;induction de l&#39;apoptose dans des cellules de mammifères après leur livraison cytosolique de la protéine formant des pores PFN 1. Récemment, le spectre des Gzms cytotoxique a été élargi de façon significative à partir de cellules de mammifères à 9,10 bactéries, ainsi qu&#39;à 11 certains parasites. En outre, les fonctions, extracellulaires non-classiques de GZMA et GZMB ainsi que la signification b...

Les Gzms sont une famille de serine proteases homologues fortement localisées dans les lysosomes spécialisées des CTL et les cellules NK 1. Les granules cytotoxiques de ces cellules tueuses contiennent également des protéines de la membrane perturbateurs PFN et GNLY qui sont libérés simultanément avec les Gzms lors de la reconnaissance d&#39;une cellule cible destiné à l&#39;élimination 2,3. Il existe cinq Gzms chez l&#39;homme (GZMA, B, H, K et M), et 10 chez des souris (Gzms GZMA - G, K, M et N). GZMA et GZMB sont les plus abondants et largement étudiés chez l&#39;homme et la souris 1. Cependant, des études plus récentes ont commencé à enquêter sur les voies de mort cellulaire ainsi que les effets biologiques supplémentaires induits par les autres, soi-disant Gzms orphelins de la santé et de la maladie 4. La fonction la plus connue des Gzms, en particulier de GZMA et GZMB, est l&#39;induction de la mort cellulaire programmée dans des cellules de mammifère lorsqu&#39;elle est remise dans les cellules cibles par PFN 5,6. Pourtant, Des études plus récentes ont également démontré des effets extracellulaires des Gzms avec un impact profond sur la réglementation et l&#39;inflammation immunitaire, indépendamment de livraison cytosolique par PFN 7,8. Le spectre de cellules qui sont tuées efficacement cytosolique après l&#39;entrée des Gzms a également été récemment élargie à partir de cellules de mammifères à des bactéries et même 9,10 11 certains parasites. Ces découvertes récentes ont ouvert un nouveau champ pour les chercheurs GZM. Par conséquent, un, à haut rendement système d&#39;expression mammalien rentable solution va considérablement faciliter la voie à ces études futures. Gzms natives humaines, de souris et de rat ont été purifiés avec succès à partir de la fraction de granulés de CTL et NK lignées cellulaires 14/12. Cependant, dans les mains, le rendement de ces techniques de purification est dans la gamme de culture inférieure à 0,1 mg / L de cellules (observation non publiée et 12). En outre, la résolution chromatographique d&#39;un seul GZM sans contamination par other Gzms et / ou des protéines qui sont également présentes dans les granules contestent (données non publiées et 12,14). Gzms recombinants ont été produites dans des bactéries, des levures 15 16, des cellules d&#39;insectes 17 et même dans des cellules de mammifères telles que HEK 293 18,19. Seuls les systèmes d&#39;expression de mammifères portent le potentiel de produire des enzymes recombinantes avec des modifications post-traductionnelles identiques à la protéine native cytotoxique. Modifications post-traductionnelles ont été impliqués avec l&#39;absorption spécifique par endocytose et la localisation intracellulaire de la protease dans les cellules cibles de 20 à 22. Par conséquent, en utilisant pHLsec 23 (un cadeau genre de Radu Aricescu et Yvonne Jones, Université d&#39;Oxford, Royaume-Uni) comme l&#39;épine dorsale de plasmide pour l&#39;expression GZM, nous avons établi un système simple, rapide et économique efficace pour la production de protéines à haut rendement dans des cellules HEK293T. pHLsec combine un activateur de CMV avec un promoteur de poulet Β-actine; Ensemble, ces éléments démonstrationsted la plus forte activité de promoteur dans diverses lignées cellulaires 24. En outre, le plasmide contient un Β-globine de lapin intron, des signaux de sécrétion et de Kozak optimisée, une 6xHis-tag-Lys et un signal poly-A. Les inserts peuvent être clonées idéalement entre le signal de sécrétion et la-6xHis-tag Lys (Figure 1) assurer une expression optimale et l&#39;efficacité de la sécrétion de protéines dépourvues appropriées domaines N-terminaux. Pour l&#39;expression des Gzms, nous avons remplacé la séquence signal de sécrétion endogène avec le signal de sécrétion fournie par le vecteur suivie par une entérokinase (EK) place (DDDDK) afin que le traitement EK activé le Gzms sécrétées (Gzms actifs commencer par la N-terminal séquence d&#39;acides aminés IIGG 25). De plus en faveur de cette méthode, les cellules HEK293T se développent rapidement en basse à prix moyen, comme milieu de Eagle modifié par Dulbecco (DMEM), et sont bien adaptés pour la méthode de transfection au phosphate de calcium 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.

Dans la section suivante, nous allons présenter une documentation complète d&#39;une préparation GZMA pour éclairer la méthode. Nous avons également purifié succès GZMB et GzmM, produisant des résultats similaires en ce qui concerne l&#39;efficacité et l&#39;activité purification. Cependant, à partir de ces préparations plus tard nous ne montrer quelques morceaux choisis de données. Préparations GZMB à partir de cellules 293T en suivant le protocole actuel ont été utilisés dans plusieurs études publi...

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 rendement élevé et Expression System coût-efficace de granzymes humain dans des cellules de mammifères

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 vues.  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. 

Vidéo: 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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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

Recherche

Biologie

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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.

Immunoprécipitation de la chromatine (ChIP) pour doser modification des histones dynamique dans l&#39;expression génique dans les cellules humaines Activé

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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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.

Absolute mesure du rendement quantique des échantillons de poudre

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

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.

Analyse de l&#39;expression de mammifères Linker-histones sous-types

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.

Visualisation des ARNm localisés dans le réticulum endoplasmique des cellules de mammifères

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.

siRNA contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

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