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. Produzione del plasmide espressione pHLsec-GZM <ol><li> Preparare RNA totale da cellule NK umane (cellule primarie preparato come al 26 o la linea delle cellule NK NK-92 che esprimono tutte le cinque Gzms umane), utilizzando un metodo di isolamento RNA adatto e invertire trascrivere con un primo filamento kit cDNA sintetizzare, a seguito della manufacturer` raccomandazioni s. Amplificare GZM cDNA usando la PCR e clonazione in pHLsec come descritto in 23 (gentile dono di Radu Aricescu e Yvonne Jones, dell&#39;Università di Oxford, Regno Unito) utilizzando i siti AGEI e KpnI (Figura 1). NOTA: Utilizzare i seguenti primer indicati nella tabella 1. </li><li> Confermare inserti corretti di sequenziamento. Espandere i plasmidi di espressione nelle cellule DH5α e purificare utilizzando un kit di isolamento plasmide privo di endotossine e seguire le istruzioni del produttore. </li><li> Risospendere i plasmidi purificati in privo di endotossine, acqua sterile ad una concentrazione di 2 mg / ml e conservare a -20 ° C untiuso l. </li></ol> 2. Espressione dei Gzms nelle cellule HEK293T <ol><li> Preparazione dei reagenti <ol><li> Preparare terreno di coltura standard. Per di Dulbecco modificato (DMEM) contenente alte concentrazioni di glucosio (25 mm), Glutamax (4 mm), piruvato di sodio (1 mm) aggiungere il 10% di siero standard fetale bovino (FCS), la penicillina (100 unità / ml), streptomicina ( 100 mg / ml). </li><li> Preparare medio trasfezione. Per mezzo di coltura senza penicillina streptomicina aggiungere 25 mg / ml clorochina (aggiungere appena il giorno della trasfezione da 1,000x scorte in PBS, conservati a -20 ° C). </li><li> Preparare mezzo privo di siero cultura: per mezzo privo di siero per HEK293 cellule aggiungono glutamina (4 mm), la penicillina (100 unità / ml), streptomicina (100 mg / ml), e di 2,5 mg / L amfotericina B. </li><li> Preparare le soluzioni descritte in Tabella 2 e filtro con un filtro da 0,45 micron prima dell&#39;uso: </li></ol></li><li> Espansione cellule HEK293T <ol><li> Crescere HEK293T cellule in 20 ml di cultomedio ure con 150 x 25 millimetri piatti di coltura dei tessuti. </li><li> Cellule Split a 80% di confluenza (split-rapporto di 1: 4, di solito ogni giorno 3 °). Cellule liberamente allegare, possono essere staccati meccanicamente senza tripsinizzazione rigorosamente pipettando su e giù. </li><li> Cellule piastra la sera prima trasfezione per dare il 60-70% di confluenza al giorno di trasfezione (densità di semina ~ 2 x 10 5 cellule / ml). Una dimensione di preparazione al solito composto da 20-25 piatti della cultura. </li></ol></li><li> Fosfato di calcio trasfezione <ol><li> 1 ora prima della trasfezione, cambiare il terreno di coltura standard per 20 ml di mezzo di trasfezione. Vicino alla fine di questo step 1 ora di incubazione, miscelare 400 mg di DNA plasmidico con 10,95 ml DDH 2 0 e 1,55 ml di 2M CaCl 2 in provette da 50 ml (1 tubo / 5 piatti). </li><li> 1 a 2 minuti prima della trasfezione, aggiungere 12,5 ml 2x HBS a 12,5 ml della soluzione di DNA-Calcium, mescolare per inversione dei tubi e incubare per 30 secondi a temperatura ambiente. </li><li> Aggiungi the composto preparato in 2.3.2 direttamente alle cellule (5 ml per piatto), goccia a goccia nel mezzo. Spruzzare uniformemente su tutta la superficie, girando il mezzo per un colore leggermente arancione. </li><li> Incubare le piastre di coltura per 7 a 11 h in un incubatore di coltura tissutale (37 ° C, 5% CO 2 in aria umidificata). Dopo l&#39;incubazione, un bel precipitato è visibile. Rimuovere il supporto transfezione, lavare accuratamente con PBS preriscaldata e aggiungere 20 ml di terreno di coltura senza siero preparati a 2.1.3. Incubare per 72 ore in un tessuto culturale incubatore. </li><li> Analizzare l&#39;efficienza di trasfezione ed espressione eseguendo un campione (~ 20 ml) del surnatante cellulare su SDS-PAGE e colorazione con Coomassie Brilliant Blue per visualizzare una banda granzyme. </li></ol></li><li> Purificazione di Gzms dalla cultura surnatante da Nickel-IMAC <ol><li> Dopo l&#39;incubazione, decantare il surnatante di colture cellulari in 250 mL e chiaro per centrifugazione. Effettuare un primo giro (400 xg, 10 min, 4 °C) che cancellerà il mezzo da cellule indipendenti. Trasferire il surnatante in freschi 250 ml provette e centrifugare a 4000 xg, 30 min a 4 ° C per rimuovere i detriti rimanenti. </li><li> Aggiungere 5 ml di 5 M NaCl, 6,25 ml di 2 M Tris-Base (pH 8) e 1 ml di 0,25 M Niso 4 per 250 ml di surnatante eliminato. Filtrare il surnatante con un gruppo filtro di vuoto 500 ml (0,45 mm). </li><li> Equilibrare una colonna Nichel-IMAC con la Sua vincolante tampone A (10 volumi di colonna, CV) utilizzando un sistema FPLC adeguato. </li><li> Applicare il surnatante eliminato alla colonna equilibrata ad un flusso di 0,5 ml / min a 4 ° C. </li><li> Dopo che è stato applicato il surnatante, lavare colonna con la sua impegnativa buffer finché assorbimento UV linea di base viene raggiunto (di solito 10 CV). </li><li> Eluire Gzms con lineare 20 ml gradiente (Da 0 a 250 mM imidazolo) ad una portata di 0,5 ml / min, mentre la raccolta 2 ml-frazioni. Analizzare le frazioni di eluizione di SDS-PAGE e colorazione Coomassie. </li></ol></li><li> Enterokinase (EK)trattamento e pulizia finale mediante cromatografia a scambio cationico <ol><li> Pool GZM contenenti frazioni in un tubo dialisi con ≤10 MWCO. Conservare un piccolo campione a -20 ° C, come il controllo pre-EK. </li><li> Aggiungere EK a 0,02 unità / 50 ml di surnatante iniziale direttamente alla frazione pool nel tubo di dialisi e dializza O / N (almeno 16 ore) a temperatura ambiente in EK-buffer (4 L, cambiamento tampone volta). </li><li> Il giorno dopo, analizzare EK trattata Gzms rispetto al pre-EK controllo da SDS-PAGE e colorazione Coomassie. </li><li> Quando l&#39;elaborazione N-terminale è completo, cambiare tampone di dialisi a S tampone A (4 L) e dializza per altri 4 ore a RT. Dializzato filtro (0,45 micron). </li><li> Equilibrare un S-colonna con tampone A. S Caricare il campione nella colonna ad una velocità di flusso di 0,5 ml / min a 4 ° C. </li><li> Dopo il caricamento del campione, lavare la colonna con tampone S A fino UV basale assorbanza viene raggiunta (circa 10 CV). Eluire le Gzms con un gradiente lineare di 30 ml (150 a 1,000 mM NaCl). GzmA eliuti a ~ 650 mM NaCl, GzmB a ~ NaCl 700 mm e GzmM a ~ NaCl 750 mm. </li><li> Analizzare le frazioni di eluizione di SDS-PAGE e test colorimetrici (vedi sotto). Pool frazioni contenenti Gzms e concentrato (circa 30 volte, ad una concentrazione di circa 100 mM) in filtri di spin (15 ml, 10 MWCO). Aliquota i preparativi GZM concentrate e conservare a -80 ° C. </li></ol></li></ol> Attività 3. Test GZM <ol><li> Preparazione dei reagenti <ol><li> Preparare colorimetrico tampone: 50 mm Tris-Base, pH 7. <ol><li> Per la misura di GzmA, aggiungere Na-CBZ-L-lisina thiobenzyl estere (S-Bzl) (BLT) al tampone ad una concentrazione finale di 0,2 mM e 5,5&#39;-ditio-bis (acido 2-nitrobenzoico) (DTNB) ad una concentrazione di 0,22 mM. Per GzmB, includere 0,2 mm DTNB mM Boc-Ala-Ala-Asp-S-Bzl (DAA) e 0,22. Per GzmM, includere 0,2 mm Suc-Ala-Ala-Pro-Leu-p-nitroanilide (PNA) (AAPL). Peptidi sintetici sono aggiunti da adeguate soluzioni madre (in DMSO, stored a -20 ° C) al buffer appena prima del dosaggio. </li></ol></li><li> Preparare tampone clivaggio: NaCl 100 mM, 50 mM Tris-Base, pH 7.5 </li></ol></li><li> Test colorimetrici <ol><li> Distribuire piccoli campioni GZM (&lt;5 ml) da frazioni di eluizione o stock concentrati nelle piastre a 96 pozzetti a fondo piatto. Includere un controllo positivo (preparati GZM testate o greggio lisati cellulari NK) e un controllo negativo (tampone solo). </li><li> Aggiungere 100 ml di tampone a ciascun pozzetto (utilizzando una pipetta multicanale). Incubare per 10 minuti a 37 ° C. Misurare OD a 405 nm in un lettore per micropiastre. </li></ol></li><li> Test Decolleté <ol><li> Per i saggi scissione utilizzando substrati purificati, co-incubare un substrato proteico (nativa o ricombinante, 100-400 nM; alcuni esempi di substrati GZM efficienti sono presentati nella sezione risultato e nella nota alla fine di questa sezione) con diluizioni seriali di un GZM (a partire da 400 nM) in tampone per varie volte.Analizzare mediante SDS-PAGE e Coomassie o macchie d&#39;argento; o mediante Western blotting utilizzando anticorpi specifici. </li><li> Per i saggi scissione utilizzando lisati cellulari, sospendere 10 6 cellule HeLa (o qualsiasi linea cellulare tumorale disponibile) in 1 ml di tampone di saggio e lyse congelando in un / bagno di ghiaccio secco etanolo e scongelamento a 37 ° C per tre volte. Rimuovere detriti cellulari mediante centrifugazione (15.000 xg per 10 min a 4 ° C). </li><li> Co-incubare il lisato liquidati con Gzms a diverse concentrazioni e tempi di cui sopra. Cleavage viene rilevata mediante immunoblotting utilizzando anticorpi appropriati. NOTA: Alcuni esempi di substrati in buona fede per la quale gli anticorpi commerciali sono disponibili: Offerta (BH3-interagente agonisti morte di dominio) e caspasi 3 spaccati da GzmB 27,28; più ribonucleoproteins nucleari eterogenei (hnRNPA1, A2 e U) spaccati da GzmA 29; citomegalovirus phosphoprotein 71 da GzmM 30. </li></ol></li></ol>

<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.

Il ruolo classico e ampiamente studiato di GzmA e GzmB è l&#39;induzione di apoptosi nelle cellule di mammifero dopo la loro consegna citosolico dal formano pori proteine ​​PFN 1. Recentemente, lo spettro citotossica delle Gzms stato ampliato significativamente da cellule di mammifero ai batteri 9,10, così come a certi parassiti 11. Inoltre, i non classici, funzioni extracellulari di GzmA e GzmB nonché il significato biologico dei vari Gzms orfani sono ancora oscuri. Pertanto, l&#...

I Gzms sono una famiglia di serina proteasi altamente omologhi localizzate nei lisosomi specializzati di CTL e cellule NK 1. I granuli citotossici di queste cellule killer contengono anche i-membrana interrompere proteine ​​PFN e GNLY che vengono rilasciati contemporaneamente alle Gzms accreditato in una cella di destinazione destinata per l&#39;eliminazione 2,3. Ci sono cinque Gzms nell&#39;uomo (GzmA, B, H, K e M), e 10 Gzms nei topi (GzmA - G, K, M e N). GzmA e GzmB sono i più abbondanti e ampiamente studiati sugli esseri umani e nei topi 1. Tuttavia, studi più recenti hanno cominciato a studiare le vie di morte cellulare e gli effetti biologici supplementari mediati dalle altre, cosiddette Gzms orfani in materia di salute e di malattia 4. La funzione più nota delle Gzms, in particolare di GzmA e GzmB, è l&#39;induzione della morte cellulare programmata in cellule di mammifero quando espresso nelle cellule bersaglio da parte PFN 5,6. Tuttavia, Studi più recenti hanno anche dimostrato effetti extracellulari dei Gzms con profondo impatto sulla regolazione immunitaria e infiammazione, indipendentemente dalla consegna citosolica da PFN 7,8. Lo spettro di cellule che vengono uccisi in modo efficace dopo l&#39;entrata citosolico dei Gzms è stato recentemente ampliato da cellule di mammifero a batteri 9,10 e anche alcuni parassiti 11. Queste scoperte recenti aperto un campo completamente nuovo per i ricercatori GZM. Pertanto, un alto rendimento del sistema di espressione mammifero economicamente efficiente faciliterà notevolmente la strada a tali studi futuri. Umani, topi e ratti Gzms nativi sono stati purificati con successo dalla frazione granello di CTL e NK cellule linee 12-14. Tuttavia, nelle nostre mani il rendimento di tali tecniche di purificazione è nella gamma di cultura inferiore a 0,1 mg / L cella (osservazioni non pubblicate e 12). Inoltre, la risoluzione cromatografica di un singolo GZM senza contaminazione da other Gzms e / o proteine ​​che sono presenti anche nei granuli è impegnativo (dati inediti e 12,14). Gzms ricombinanti sono state prodotte in batteri, lieviti 15 16, cellule di insetto 17 e in anche in cellule di mammifero, come HEK 293 18,19. Solo i sistemi di espressione di mammifero portano il potenziale di produrre enzimi ricombinanti con modificazioni post-identico alla proteina citotossica nativo. Modificazioni post-traduzionali sono stati implicati con l&#39;assorbimento specifico per endocitosi e la localizzazione intracellulare della proteasi all&#39;interno delle cellule bersaglio 20-22. Pertanto, utilizzando pHLsec 23 (un gentile dono di Radu Aricescu e Yvonne Jones, dell&#39;Università di Oxford, Regno Unito) come spina dorsale plasmide per l&#39;espressione GZM, abbiamo istituito un sistema semplice, tempo e conveniente per alto rendimento produzione di proteine ​​in HEK293T cellule. pHLsec combina un esaltatore di CMV con un pollo Β-actina promotore; insieme, questi elementi dimoted il più forte promotore attività in varie linee cellulari 24. Inoltre, il plasmide contiene un coniglio Β-globina introne, segnali Kozak e secrezione ottimizzati, a-6xHis-tag Lys e un poli-A segnale. Gli inserti possono essere clonate convenientemente tra il segnale di secrezione e Lys-6xHis-tag (Figura 1) assicurando espressione ottimale e efficienza secrezione di proteine ​​prive appropriate domini N-terminali. Per l&#39;espressione delle Gzms, abbiamo sostituito la sequenza segnale di secrezione endogena con il segnale di secrezione fornito dal vettore seguito da un enterokinase (EK) sito (DDDDK) in modo che il trattamento EK attivato il Gzms secrete (Gzms attivi iniziare con N-terminale sequenza aminoacidica IIGG 25). Inoltre, a favore di questo metodo, le cellule HEK293T crescono rapidamente in basso prezzo medio, come Dulbecco Modified mezzo di Eagle (DMEM), e sono adatti per il calcio-fosfato metodo trasfezione conveniente.

<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.

Nella seguente sezione presenteremo una documentazione completa di un preparato GzmA per illuminare il metodo. Abbiamo anche con successo purificati GzmB e GzmM, producendo risultati simili in materia di efficienza e l&#39;attività di depurazione. Tuttavia, da tali preparati più tardi mostreremo solo pochi pezzi selezionati di dati. Preparazioni GzmB dalle cellule 293T seguenti l&#39;attuale protocollo sono stati utilizzati in diversi studi pubblicati, mettendo in evidenza la loro attività in diversi sistemi di dosag...

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.

A High Yield e Cost-efficient Sistema Espressione di granzimi umane in cellule di mammifero

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 Views.  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.

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

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

Ricerca

Biologia

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

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

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

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

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

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.

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell&#39;espressione genica attivata nelle cellule umane

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.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

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 Quantum la misurazione del rendimento dei campioni di polvere

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.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

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.

Analisi di espressione di mammifero Linker-istoni Sottotipi

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.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

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 screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

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