Questo progetto è stato finanziato dal Fondo Sociale Europeo in Baden-Wuerttemberg, Germania.

1. Aumento di plasmidi contenenti richiesto codifica sequenze di DNA (CDS) <ol><li> Medio pre-SOC calda, che è incluso nel kit di trasformazione, a temperatura ambiente e le piastre di agar LB contenenti 100 ug / ml di ampicillina a 37 ° C. Equilibrare il bagno d&#39;acqua a 42 ° C. </li><li> Scongelare un flacone di chimicamente componente E. coli su ghiaccio. </li><li> Aggiungere 1-5 ml di plasmide (10 pg a 100 ng) contenente i CDS nel flaconcino di chimicamente componente E. coli e mescolare delicatamente. Non mescolare le cellule pipettando. Dopo aver aggiunto plasmidi, Mescolare agitando delicatamente il tubo. </li><li> Incubare la miscela in ghiaccio per 30 min. </li><li> Heat-shock E. coli per 30 sec a 42 ° C in un bagno d&#39;acqua senza agitazione. </li><li> Posizionare la fiala in ghiaccio per 2 min. </li><li> Aggiungere 250 microlitri di media SOC pre-riscaldato a E. coli e agitare i batteri orizzontalmente a 300 rpm per 1 ora a 37 ° C in un incubatore batterica. </li><li><html> Distribuire 100 ml e 150 ml di trasformazione mix su piastre di agar LB pre-riscaldato, capovolgere le piastre ed incubare per una notte a 37 ° C. </li><li> Dopo colonie diventano visibili, inoculare una singola colonia di ciascuna piastra in una provetta da 15 ml contenente 5 ml di terreno LB con 100 ug / ml di ampicillina. Incubare durante la notte a 37 ° C con agitazione a 300 rpm fino a quando la cultura è alla fine del registro o fase stazionaria. </li><li> Per la conservazione a lungo termine di trasformare E. coli, pipetta 75 ml di 100% glicerolo nel esageratamente. Aggiungere 225 ml di coltura batterica (magazzino congelato conterrà 25% glicerolo), mescolare bene e conservare il esageratamente a -80 ° C. </li><li> Isolare i plasmidi contenenti i CDS richiesti utilizzando un kit di purificazione plasmide secondo le istruzioni del produttore. </li><li> Determinare la concentrazione plasmide utilizzando uno spettrofotometro. </li><li> Preparare aliquote di 20 ml e conservare a -20 ° C per un tempo prolungato. </li></ol>_title &quot;&gt; 2. Amplificazione dei plasmidi Inserti e aggiunta di Poly T-tail da Polymerase Chain Reaction (PCR) <ol><li> NOTA: Per ottenere il DNA stampo per la trascrizione in vitro (IVT), il CDS di interesse, qui eGFP, viene amplificato mediante PCR. Contemporaneamente, un poli T-coda 120 thymidines (T) viene aggiunto l&#39;inserto utilizzando un primer reverse con T 120 estensione. In tal modo, gli mRNA generati ottenere un poli A-coda con una lunghezza fissa dopo l&#39;IVT. </li><li> Preparare la miscela di PCR come indicato nella tabella 1. </li><li> Mescolare accuratamente la miscela di reazione. NOTA: prodotto della PCR può essere utilizzato in 4-5 reazioni IVT. Per aumentare la quantità di DNA stampo FPI, il volume totale della miscela PCR può essere aumentata. Diversi provette PCR contenenti ciascuna 100 microlitri miscela di PCR possono essere utilizzati per ottenere una resa sufficiente di prodotto di PCR. </li><li> Mettere le provette per PCR in un termociclatore ed eseguire il PCR utilizzando il protocollo ciclismo PCR (Tabella 2). </li><li> Pulire il PReazione CR utilizzando un kit di purificazione PCR secondo le istruzioni del produttore ed eluire il DNA con 20 di acqua priva di nucleasi microlitri. </li><li> Misurare la concentrazione del DNA usando uno spettrofotometro. NOTA: La concentrazione di DNA rilevato è di circa 300 mg / ml. Pertanto, l&#39;importo complessivo previsto dovrebbe essere ~ 6 mg. La quantità di DNA per altre proteine ​​possono differire a seconda della lunghezza dei CDS, quantità di plasmide usato per la PCR, temperatura di appaiamento dei primer, e il numero di cicli. </li><li> Congelare il DNA a -20 ° C per un lungo periodo o utilizzare direttamente per IVT. </li></ol> 3. Controllo Passo: Qualità della PCR prodotto <ol><li> Controllare la qualità del prodotto di PCR mediante elettroforesi su gel di DNA. </li><li> Miscelare la quantità desiderata di agarosio con 1x TBE in un pallone. Per un gel di agarosio all&#39;1%, aggiungere 0,5 g di agarosio a 50 ml di TBE 1x. </li><li> Forno a microonde fino l&#39;agarosio è sciolto. Non lasciate bollire. Assicurarsi che l&#39;agarosio è completamente in soluzione. </li><li> Aggiungere 5 ml macchia gel di acido nucleico a 50 ml di gel di agarosio. </li><li> Versare il gel in una scatola di gel di agarosio, impostare il pettine, e attendere circa 1 ora fino a quando il gel solidificato. </li><li> Rimuovere il pettine e posizionare la scatola gel in una vaschetta per gel. </li><li> Mescolare 2 ml (200 ng) di 80-10,000 bp DNA-Ladder e 200 ng di prodotto di PCR ciascuna con 2 ml di tampone di caricamento 6x in un volume totale di 12 ml. </li><li> Caricare con cautela campioni di DNA nei pozzetti del gel di agarosio. </li><li> Utilizzando TBE 1x come tampone di corsa, eseguire il gel di agarosio a 100 V per 1 ora. </li><li> Visualizzare le bande di DNA su un sistema di imaging (Figura 2). </li></ol> 4. Trascrizione in vitro (IVT) <ol><li> NOTA: Dopo la PCR, gli inserti plasmide sono amplificati e viene aggiunto un poli T-coda. Durante la IVT, informazione genetica è trascritto dal DNA all&#39;RNA. L&#39;mRNA trascritto generato viene utilizzato per produrre proteine ​​nelle cellule. </li><li> Pulire l&#39;area di lavoro e le pipette con RNasi dsoluzione di decontaminazione. Usa PCR punte pulito e sterile a doppio filtro per pipette e cambiare frequentemente i guanti per minimizzare la contaminazione di reazione si mescola con RNasi. </li><li> Preparare la miscela analogico NTP / cap come descritto nella Tabella 3. </li><li> Mescolare la miscela analogico NTP / cap accuratamente su vortex e centrifugare brevemente. </li><li> Montare la miscela di reazione IVT, come descritto nella tabella 4. </li><li> Mescolare la miscela di reazione IVT accuratamente pipettando gentilmente su e giù. Poi centrifugare la provetta PCR brevemente per raccogliere la miscela sul fondo della provetta. </li><li> Incubare a 37 ° C per 3 ore in un thermomixer. NOTA: Il tempo di incubazione ottimale dipende dalla lunghezza degli inserti e l&#39;efficienza trascrizionale dato modello. Per inserti brevi (&lt;500 nt), un tempo di incubazione più lungo può essere vantaggioso. Un esperimento temporale corso può essere fatto per determinare il tempo ottimale di incubazione per il massimo rendimento. Pertanto, impostare le reazioni FPI, per example, per 1, 2, 3, 4, 6 ore, e notte di incubazione e determinare la quantità di mRNA. La cinetica della trascrizione in vitro per la generazione di eGFP mRNA è illustrato nella figura 3. </li><li> Per rimuovere il DNA stampo, aggiungere 1 ml di DNasi (2 U / mL) alla miscela di reazione IVT, mescolare bene e incubare 15 minuti a 37 ° C. </li><li> Purificare la miscela di reazione utilizzando un kit di purificazione RNA secondo le istruzioni del fabbricante. Eluire l&#39;mRNA modificato dalla membrana Spin Column due volte con 40 di acqua priva di nucleasi microlitri. NOTA: La concentrazione di RNA rilevato è di circa 1.500 mcg / ml. Pertanto, l&#39;importo complessivo previsto dovrebbe essere ~ 120 mg. La quantità di mRNA sintetizzato per altre proteine ​​possono differire a seconda della lunghezza del CDS e la quantità di prodotto di PCR utilizzati per IVT. </li></ol> 5. Il trattamento di mRNA purificato con Antartico fosfatasi <ol><li> NOTA: L&#39;mRNA generata viene trattato con fosfatasi per rimuovere 5 &#39;trifosfati, che possono essere riconosciuti da RIG-I e portano alla attivazione immunitaria. Inoltre, il trattamento con fosfatasi evita re-circularization in una reazione auto-ligazione. </li><li> Aggiungere 9 ml di tampone di reazione 10x fosfatasi Antartico a 79 ml di soluzione di mRNA purificata. Successivamente, aggiungere 2 ml di fosfatasi Antartico (5 U / ml) e mescolare delicatamente il campione. Incubare la miscela di reazione a 37 ° C per 30 min. </li><li> Purificare la miscela di reazione utilizzando un kit di purificazione RNA secondo le istruzioni del fabbricante. Eluire l&#39;mRNA modificato dalla membrana Spin Column due volte con 50 di acqua priva di nucleasi microlitri. </li><li> Misurare la concentrazione degli mRNA modificati utilizzando uno spettrofotometro. Verificare che il rapporto di assorbanza a 260 nm / 280 nm (A 260 / A 280) è ≥ 1,8 e il rapporto A 260 / A 230 è 2,0, che indica la purezza. NOTA: Il rendimento totale atteso dovrebbe essere di circa 90 mg, che è sufficient per circa 36 trasfezioni mRNA. </li><li> Regolare la concentrazione di mRNA di 100 ng / ml per aggiunta di acqua priva di nucleasi. </li><li> Aliquota della mRNA modificato in monouso aliquote necessarie per transfezioni e conservare a -80 ° C. Evitare ripetuti cicli di congelamento e scongelamento. Adeguatamente preparati e conservati mRNA è stabile per molti anni. Durante il lavoro, tenere sempre l&#39;mRNA modificato sul ghiaccio. </li></ol> 6. Controllo Passo: Qualità di mRNA sintetizzato Modificato <ol><li> Controllare la qualità del mRNA modificato mediante elettroforesi su gel di RNA. </li><li> Miscelare la quantità desiderata di agarosio con 1x TBE in un pallone. Per un gel di agarosio all&#39;1%, aggiungere 0,5 g di agarosio a 50 ml di TBE 1x. </li><li> Forno a microonde fino l&#39;agarosio è sciolto. Non lasciate bollire. Assicurarsi che l&#39;agarosio è completamente in soluzione. </li><li> Aggiungere 5 ml macchia gel di acido nucleico a 50 ml di gel di agarosio. </li><li> Versare il gel in una scatola gel, impostare il pettine, e attendere circa 1 ora fino a quando il gel è solisolidificati. </li><li> Rimuovere il pettine e posizionare la scatola gel in una vaschetta per gel. </li><li> Mescolare 3 microlitri (3 mg) di 0,5-10 kb RNA Ladder e 200 ng di sintetizzati modificato mRNA ciascuna con 7 ml di tampone di caricamento in un volume totale di 10 microlitri. La composizione del tampone di caricamento sono descritte nella Tabella 5. </li><li> Denaturare la scala e campioni di mRNA a 70 ° C per 10 min. </li><li> Sistemare con cura la scala ed i campioni di mRNA sul gel di agarosio 1%. </li><li> Eseguire elettroforesi a 100 V per 1 ora con TBE 1x come tampone di corsa. </li><li> Visualizzare la scala e bande di mRNA su un transilluminatore UV e fotografia mediante un sistema di documentazione gel (Figura 4). </li></ol> 7. Preparazione delle cellule per la trasfezione <ol><li> Piastra 2 x 10 5 cellule (cellule HEK293) per pozzetto della piastra 12 pozzetti. </li><li> Incubare le cellule per una notte a 37 ° C in un incubatore cella. NOTA: Il giorno della trasfezione, le cellule devono avere reazionehed 80-90% di confluenza. </li></ol> 8. L&#39;esecuzione di mRNA trasfezione di cellule <ol><li> Scongelare il mRNA modificato. </li><li> Generare i lipoplexes per trasfezione. </li><li> Aggiungere 25 ml (2,5 mg) di mRNA modificato e 2 ml di reagente di lipide cationico trasfezione a 473 microlitri Opti-MEM (mezzo essenziale minimo) ho ridotto medio siero per generare la miscela di trasfezione per la trasfezione di un pozzetto di una piastra 12 pozzetti. Scala i volumi in base al numero di pozzetti da transfettate. Come controllo negativo, preparare una miscela di trasfezione, senza mRNA. </li><li> Miscelare i componenti delicatamente con una pipetta. Incubare la miscela di trasfezione a temperatura ambiente per 20 min per generare lipoplexes per trasfezione. NOTA: La miscela di trasfezione non contiene antibiotici. Quindi, fare attenzione a garantire la sterilità durante la manipolazione delle cellule. </li><li> Lavare le cellule con 500 DPBS microlitri / bene. </li><li> Aggiungere 500 microlitri miscela di trasfezione di un pozzetto di un pla 12 pozzettite. </li><li> Incubare le cellule per 4 ore a 37 ° C e 5% di CO 2. </li><li> Aspirare la miscela di trasfezione e aggiungere 1 ml di mezzo completo di coltura cellulare per le cellule. </li><li> Incubare le cellule per 24 ore nell&#39;incubatrice cellulare. </li><li> Eseguire fluorescenza microscopica analisi usando un microscopio a fluorescenza (Figura 5). NOTA: Per esaminare l&#39;espressione di altre proteine ​​nelle cellule, che sono prodotti da trasfezione con altri mRNA che codifica eGFP mRNA, le cellule possono essere coltivate su vetrini. Poi la trasfezione può essere eseguito e le cellule può essere trattata con anticorpi appropriati e analizzato al microscopio a fluorescenza. </li></ol> 9. Analisi di citometria a flusso eGFP espressione in cellule <ol><li> Rimuovere terreno di coltura cellulare dai pozzetti e lavare i pozzetti con 1 ml DPBS (senza calcio e magnesio). Aggiungere 500 microlitri tripsina / EDTA per pozzetto per staccare le cellule dalla superficie delle piastre di coltura cellulare. Successivamente, aggiungere 500TNS ml per pozzetto per inattivare la tripsina. </li><li> Centrifugare le cellule staccate per 5 min a 300 xg a temperatura ambiente. Rimuovere il surnatante lasciando solo il pellet. Risospendere il pellet di cellule in 150 microlitri soluzione di fissazione delle cellule e trasferire la sospensione cellulare in un tubo di citometria a flusso. </li><li> Analizzare la percentuale di cellule che esprimono eGFP e l&#39;intensità di fluorescenza utilizzando Geo medio (media geometrica dell&#39;intensità di fluorescenza) (Figura 6). </li></ol> 10. Misura di espressione della proteina nel tempo <ol><li> Eseguire mRNA trasfezione di cellule in 3 pozzetti di una piastra a 12 pozzetti come descritto nel passaggio 8. Inoltre, incubare 3 pozzetti di cellule HEK293 con la miscela di trasfezione senza l&#39;aggiunta di mRNA. </li><li> Misurare l&#39;espressione di eGFP 1, 2, e 3 giorni dopo la trasfezione per valutare la durata di espressione proteica (Figura 7). </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

La terapia mRNA ha un enorme potenziale nel campo della medicina rigenerativa, il trattamento di malattie e vaccinazione. In questo video, dimostriamo la produzione di un mRNA modificato stabilizzata per l&#39;induzione dell&#39;espressione proteica nelle cellule. Usando questo protocollo, altri mRNA desiderati possono essere generati. La sintesi in vitro di mRNA modificato consente trasfezione di cellule con mRNA desiderati per indurre l&#39;espressione di proteine ​​bersaglio. In tal modo, la proteina des...

Nelle cellule, la trascrizione di RNA messaggero (mRNA) e la seguente traduzione di mRNA in proteine ​​desiderate assicurare il corretto funzionamento delle cellule. Malattie genetiche ereditarie o acquisite possono portare a insufficiente e disfunzionale sintesi delle proteine ​​e causare gravi malattie. Pertanto, un nuovo approccio terapeutico per indurre la produzione di proteine ​​mancanti o difettosi è la consegna esogena di sintetica modificata mRNA in cellule, che codifica per la proteina desiderata. In tal modo, le cellule vengono attivate per sintetizzare le proteine ​​funzionali, che normalmente non possono produrre o sarebbe naturalmente non hanno bisogno. Usando questo approccio, malattie genetiche possono essere corretti dalla introduzione di mRNA che codifica per la proteina difettosa o mancante 1. La terapia mRNA può essere utilizzato anche per la vaccinazione di sintetizzare antigeni proteici che sono espressi dalle cellule tumorali o patogeni. In tal modo, il sistema immunitario dell&#39;ospite può essere attivato per eliminare efficacemente le cellule tumorali o impedire infeZIONI 2,3. Inoltre, negli ultimi anni, mRNA è stato usato con successo per generare cellule staminali pluripotenti indotte (iPSCs). A tal fine, fibroblasti sono state trasfettate con mRNA di indurre l&#39;espressione di fattori di riprogrammazione 4-6 e convertirli in iPSCs con un enorme potenziale in medicina rigenerativa. In precedenza, l&#39;uso di mRNA convenzionale è stata associata con scarsa stabilità e forte immunogenicità. In questo modo, le applicazioni cliniche di mRNA convenzionali erano limitate. Tuttavia, la sostituzione di citidina e uridina da 5-methylcytidine e pseudouridina all&#39;interno della molecola di mRNA mediante Kariko e colleghi reso molecole di mRNA stabili nei fluidi biologici e drammaticamente ridotto attivazione immunitaria 7-10, che ora permette l&#39;applicabilità clinica di mRNA modificati. Utilizzando mRNA modificati sinteticamente prodotti, le trascrizioni del gene desiderato può essere recapitato temporaneamente in vivo 11o in vitro per indurre l&#39;espressione della proteina. L&#39;mRNA introdotto è tradotto in condizioni fisiologiche dalla macchina di traduzione cellulare. A causa della mancanza di integrazione nel genoma della cellula ospite, rispetto ai vettori di terapia genica virale, il rischio di oncogenesi è impedito 12,13. Pertanto, la terapia con mRNA sintetico modificato andrà meglio accettazione clinica nel futuro. Qui, descriviamo un protocollo dettagliato per la produzione di mRNA modificato (Figura 1), trasfezione delle cellule con mRNA e la valutazione di espressione della proteina in cellule trasfettate.

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:172px;">Catalog Number</td>
			<td style="width:435px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell culture</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose</td>
			<td>PAA</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Life Technologies</td>
			<td>10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:157px;">PAA</td>
			<td>P11-010</td>
			<td>100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td>PAA</td>
			<td>M11-004</td>
			<td>200 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium&#160;</td>
			<td>PAA</td>
			<td>E15-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.04% Trypsin / 0.03% EDTA&#160;</td>
			<td>Promocell</td>
			<td>C-41020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TNS</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td style="width:435px;">Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-293 cells</td>
			<td style="width:157px;">ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates, 12-well&#160;</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture flask (75 cm2)&#160;</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&#160;</td>
			<td>Eppendorf</td>
			<td>0030 121.589</td>
			<td>Safe-Lock, Biopur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes&#160;</td>
			<td>greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:295px;">PCR clean and sterile epT.I.P.S. dualfilter pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;">10 &#181;l: 022491202,&#160;&#160;&#160; 100 &#181;l: 022491237, 1000 &#181;l: 022491253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryovial</td>
			<td>greiner bio-one</td>
			<td>122279-128</td>
			<td>Cryo.s&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">14 ml polypropylene round bottom tube for bacterial culture&#160;</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid amplification and purification</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA 3.3_eGFP Plasmid&#160;</td>
			<td style="width:157px;">Addgene</td>
			<td>26822</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">One Shot Top10 chemically component Escherichia coli&#160;</td>
			<td>Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile water (Ampuwa)</td>
			<td>Fresenius Kabi</td>
			<td>1636071</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB medium (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X968.1</td>
			<td>Dissolve 25 g l-1 in sterile water.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB agar (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X969.1</td>
			<td>Dissolve 40 g l-1 in sterile water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin Ready Made Solution</td>
			<td>Sigma Aldrich</td>
			<td>A5354</td>
			<td>100 mg/ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mRNA production</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStar HiFidelity Polymerase Kit&#160;</td>
			<td>Qiagen</td>
			<td>202602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Kit&#160;</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Methylcytidine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1014</td>
			<td>5-Methyl-CTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pseudouridine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1019</td>
			<td>Pseudo-UTP</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:295px;">3&#180;-O-Me-m7G(5&#180;)ppp(5`)G RNA cap structure analog&#160;</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RiboLock RNase Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>40 U/&#181;l&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td>2 U/&#181;l (from MEGAscript T7 Kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic phosphatase&#160;</td>
			<td>New England Biolabs</td>
			<td>MO289S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap solution&#160;</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfection</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Media&#160;</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:435px;">Improved Minimal Essential Medium (MEM) with&#160; reduced Fetal Bovine Serum (FBS) supplementation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel electrophoresis</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelred Nucleic Acid Gel Stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>10,000x in water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peqGOLD Range Mix DNA-Ladder&#160;</td>
			<td>Peqlab</td>
			<td>25-2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5-10 kb RNA Ladder&#160;</td>
			<td>Invitrogen</td>
			<td>15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x TBE buffer</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometry analyses</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellFIX (1x)</td>
			<td>BD Biosciences</td>
			<td>340181</td>
			<td>10x concentrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Primer for insert amplification and&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; poly (T) tail PCR</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;-TTGGACCCTCGTACAGAAGCTAATACG-3&#180;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Reverse Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;- T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3&#180;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell incubator</td>
			<td>Binder</td>
			<td style="width:172px;"></td>
			<td>CO2 (5%) and O2 (20%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY cell counter&#160;</td>
			<td>Sch&#228;rfe System</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Sterile workbench&#160;</td>
			<td style="width:157px;">BDK Luft-und Reinraumtecknik GmbH</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial incubator&#160;</td>
			<td>Incutec</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ScanDrop spectrophotometer&#160;</td>
			<td>Analytic Jena</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermocycler&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>peqlab</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus for electrophoresis&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel documentation system&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACScan System&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase-contrast microscope&#160;</td>
			<td>Zeiss</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro synthesis of modified mrna for induction of protein expression in human cells

La consegna esogena di codifica messenger RNA sintetico (mRNA) per l&#39;induzione della sintesi proteica nelle cellule desiderate ha un enorme potenziale nel campo della medicina rigenerativa, biologia cellulare di base, il trattamento di malattie e riprogrammazione delle cellule. Qui, descriviamo un passo per passo protocollo per la produzione di mRNA modificato con ridotto potenziale di attivazione immunitaria e una maggiore stabilità, controllo di qualità di prodotti mRNA, trasfezione di cellule con mRNA e la verifica della espressione proteica indotta mediante citometria a flusso. Fino a 3 giorni dopo una singola trasfezione con eGFP mRNA, le cellule HEK293 trasfettate producono eGFP. In questo articolo video, la sintesi di mRNA eGFP è descritto come esempio. Tuttavia, la procedura può essere applicata per la produzione di altri mRNA desiderati. Utilizzando il sintetico modificato mRNA, le cellule possono essere indotte ad esprimere transitoriamente le proteine ​​desiderate, che normalmente non avrebbero esprimere.

Utilizzando una pcDNA 3.3 plasmide contenente il CDS di eGFP, è stato stabilito la sintesi di mRNA modificato eGFP (Figura 1). Dopo l&#39;inserimento di amplificazione mediante PCR e poli T-tailing, una banda chiara con una lunghezza di circa 1.100 bp viene rilevata (Figura 2). Aumentando il tempo IVT aumentata la resa di mRNA (Figura 3). Dopo l&#39;IVT, una fascia mRNA chiaro con una lunghezza di circa 1.100 bp è stato rilevato, che corrisponde alla lunghezza di eGFP...

Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In questo articolo video, si descrive la sintesi in vitro di mRNA modificata per l&#39;induzione dell&#39;espressione proteica nelle cellule.

In vitro Sintesi di mRNA modificata per l&#39;induzione di espressione della proteina nelle cellule umane

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

in-vitro-synthesis-modified-mrna-for-induction-protein-expression

Research

JoVE Journal

Biology

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

24.8K Views.  University Hospital Tuebingen.  In questo articolo video, si descrive la sintesi in vitro di mRNA modificata per l'induzione dell'espressione proteica nelle cellule. 

Video: In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Ricerca

Biologia

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Ricostituzione in vitro di Active T. castaneum telomerasi

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling cellule staminali embrionali umane individuale mediante RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

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

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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 ...