We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

NOTA: La portata di tali sistemi di dosaggio dipende dalla disposizione apparecchiature di laboratorio HTS. Questo protocollo è facilitato da una EVO 200 robot Tecan Freedom, che svolge gestione dei liquidi in formato 96-bene. La miniaturizzazione a 384 pozzetti è anche possibile. Il sistema di gestione dei liquidi robotizzato è posizionata sotto una cappa a flusso laminare per mantenere condizioni di asetticità durante tutte le fasi sperimentali. Se no automazione-liquido trattamento è disponibile, il protocollo può essere facilmente adattato in formato a bassa velocità. Screening primario 1. Giorno 1: Preparare e Celle Seed NOTA: cellule seme di resa 80% di confluenza al momento di saggiare l&#39;attività luciferasica, cioè 48 ore dopo la semina. <ol><li> Risospendere tripsinizzate cellule di neuroblastoma CHP134-mycn3UTR ad una concentrazione di 2 x 10 5 cellule / ml in RPMI contenente 10% FBS e 1% di L-glutammina. Per 27 piastre preparare almeno 250 ml di<html> sospensione cellulare tenendo conto di gestione dei liquidi, Trogoli volumi morti e fasi di pipettamento ripetuti. Versare la sospensione cellulare nel serbatoio sterile e posizionarlo sulla scrivania robot. </li><li> Collocare 9 piastre a 96 pozzetti a barre bianche a fondo piatto e una scatola di 200 microlitri puntali sterili sulla scrivania robot. Mescolare la sospensione cellulare pipettando prima di ogni passo dispensa per evitare cellule sedimentazione per gravità. Dispensare 75 ml di cellule in ogni pozzetto di 9 dischi per ottenere una densità finale di 1,5 × 10 4 cellule per bene. </li><li> Coprire le piastre con i coperchi e lasciarli all&#39;esterno del incubatore per 30 minuti per permettere alle cellule di sedimentare sul fondo del pozzo. Questo ridurrà al minimo gli effetti di bordo. </li><li> Ripetere le fasi 1.2 e 1.3 due volte per preparare un numero totale di 27 piastre. </li><li> Porre le piastre a 37 ° C e 5% di CO 2 e incubare per 24 ore. </li></ol> 2. Giorno 2: Trattare le Celle con Biblioteca Compounds S copi &quot;&gt; NOTA: La piccola biblioteca molecola di Spectrum Collection si compone di 2.000 composti disposti in 8 righe e 10 colonne in 25 piastre da 96 pozzetti alla concentrazione di 10 mM in DMSO I pozzi del primo e ultime colonne vengono lasciati vuoti per i controlli. test di screening. Compound sono tipicamente eseguiti a 1-10 micron concentrazione del composto 8. Il protocollo di screening descritto qui corregge 2 micron come la concentrazione di lavoro e 24 ore come il punto finale del test. <ol><li> Scongelare 9 piastre biblioteca composto a temperatura ambiente. </li><li> Preparare due vasche con PBS sterile e soluzioni% DMSO-0.5 PBS e metterli sulla scrivania robot. Per ogni piastra biblioteca, preparare e etichettare conseguenza una piastra di diluizione - un polipropilene U-bottom piastra a 96 pozzetti con un volume di lavoro di almeno 400 microlitri. Riempire ogni pozzetto di colonne da 2 a 11 piastre di diluizione con 398 ml di PBS sterile con punte fisse del conduttore liquido. Fill colonne 1 e 12, con 50 ml di PBS sterilecon 0,5% DMSO per riprodurre la concentrazione veicolo nei pozzetti di controllo (0,02%). </li><li> Mettere due piastre di libreria, le piastre di diluizione corrispondentemente etichettati, due scatole di 50 microlitri puntali sterili e sei celle piastre sulla scrivania robot. Scoprire cellule piatti. </li><li> Dispensare 2 ml di soluzione di MM 10 stock da una delle due piastre di libreria nella piastra di diluizione corrispondente e mescolare bene. Questo si tradurrà in una concentrazione intermedia composti di 50 pM. Utilizzando lo stesso set di punte, a meno di 3 ml di diluizione 50 micron di 3 piastre da 96 pozzetti bianche codice a barre con le cellule. Questa diluizione risultati schema a 2 micron concentrazione di ogni lavoro testati composto. NOTA: Per evitare manipolazioni inutili con le punte, utilizzare un set completo di 96 consigli fin dall&#39;inizio, anche se i piatti della biblioteca contengono solo 80 composti. Questo è possibile poiché il primo e l&#39;ultimo colonne delle piastre di libreria sono vuoti. </li><li> Sostituire i puntali e ripetere Step 2.4 per la seconda piastra libreria. </li><li> Coprire le piastre cellule e restituirli per l&#39;incubatore per 24 ore. </li><li> Ripetere i passaggi 2,3-2,6 altre due volte per le piastre di libreria rimanenti. </li></ol> 3. Giorno 3: Eseguire Luciferase Assay NOTA: Prima di eseguire il saggio luciferasi, è possibile multiplex con un test di vitalità cellulare basato sulla riduzione dei resazurina per ottenere un indice di vitalità cellulare da ciascun pozzetto 9. Multiplexing luciferasi e dosaggio redditività saggi determina una riduzione del segnale di luciferasi che, tuttavia, può essere ignorato se le cellule forniscono livello sufficientemente alto di attività luciferasica. Attività luciferasi può essere rilevato da una varietà di 10,11 luciferasi reagenti commerciali e fatti in casa con segnale stabile luminescenti. <ol><li> Equilibrare il reagente ricostituito luciferasi di scelta a temperatura ambiente. Assicurarsi che il volume è sufficiente per tutti i piatti analizzati. Per 27 piastras 170 ml di reattivo luciferasi sarà richiesto tenendo conto di gestione dei liquidi, Trogoli volume morto e fasi di pipettamento ripetuti. Versare il reagente luciferasi in un serbatoio e posizionarlo sulla scrivania robot. </li><li> Rimuovere 9 piatti con le cellule dal termostato, metterli sulla scrivania robot, scoprire e attendere fino riportati a temperatura ambiente. Aggiungere 50 ml di reagente luciferasi per piastra bene da piatto. Tra le piastre, introdurre un ritardo pari al tempo di lettura luminescenza di una piastra. Ciò assicurerà che tutte le piastre vengono misurate entro l&#39;intervallo di tempo pari dell&#39;aggiunta del reagente. </li><li> Dopo 30 min di incubazione, posizionare il primo piatto in luminometro e misurare luminescenza dopo una breve fase di agitazione. Ripetere l&#39;operazione per tutte le piastre. Registrare il codice a barre di ciascuna piastra e associarlo al file di dati corrispondente per evitare errori derivanti da un gran numero di piatti trattati. </li><li> Ripetere i passaggi 3,2-3,3 due volte per i restanti 18 piatti con le cellule. </li&gt; <li> Raccogliere il residuo reattivo luciferasi e conservarlo a -20 ° C. </li></ol> 4. Giorno 4: Analisi dei dati <ol><li> Processo dati sperimentali utilizzando la normalizzazione di tutti i campioni alla media di sulla piastra controlli trattati con veicolo. Per ogni composto biblioteca, calcolare il valore medio X e SD su triplica. </li><li> Selezionare up-regolazione e down-regolazione colpi fissando una soglia di X ± k × SD, dove la X valore medio e deviazione standard vengono calcolate su tutti i valori di analisi elaborati, e k è una costante definita. </li><li> Selezionare una soglia arbitraria cut-off &lt;20% dei controlli trattati con veicolo quando una riduzione del segnale di luciferasi è probabilmente associata a tossicità composto. Scartando i colpi di sotto di questa soglia ridurrà notevolmente il numero di colpi falsi positivi del contro-screening. NOTA: Invece di normalizzazione a base di controllo, dati sperimentali possono essere elaborati applicando Z punteggio o la sua robusta analogico B snucleo 12. Inoltre, approcci statistici alternativi per la selezione hit sono disponibili 12. </li></ol> 5. Counter-proiezione NOTA: composti identificati nella schermata principale (classificati come &#39;hits&#39;) sono confermate e valutate per la specificità da un contro-screening. <ol><li> Cherry raccogliere i successi selezionati dalle aliquote conservato in provette con codice a barre 2D singoli e creare nuovo &quot;hit piatti&quot;, salvare il layout corrispondente. Non dimenticate di lasciare posizioni libere per i controlli. NOTA: In mancanza di un efficiente sistema di cherry-picking, si potrebbe verificare sia il controllo e 3 &#39;UTR cellule-cuscinetto in parallelo nel screening primario. In questo caso, specifica selezione di composti con effetti dipendenti solo sulla introdotta 3 &#39;UTR sarà possibile dopo la proiezione primaria eliminando la necessità di contro-screening. Tuttavia, la proiezione parallelo due linee cellulari raddoppierà dosaggioi costi. </li><li> Seguendo il protocollo per lo screening primario (sezione &quot;Giorno 1: Preparare e Cells Seed&quot;), per ogni &quot;hit piastra&quot; preparare tre piastre da 96 pozzetti di ciascuna CHP134-mycn3UTR e CHP134-CTRL cellule stabili. </li><li> Seguendo il protocollo per lo screening primario (sezione &quot;Giorno 2: Trattare celle con Biblioteca Compounds&quot;), utilizzare ogni piastra colpo per il trattamento di tre CHP134-mycn3UTR e tre piastre CHP134-CTRL a 2 micron concentrazione di lavoro. NOTA: Mentre il contatore schermo qui descritto viene eseguito in triplicato alla dose singola di 2 mM, potrebbe essere vantaggioso per sostituire i replicati standard e verificare i risultati in 3 concentrazioni diluizioni di 10 volte da 2 mM a 20 nM. L&#39;applicazione di questo approccio non solo consente di confermare i dati della schermata principale, ma anche di ottenere dati preliminari dose-risposta sui risultati primari, minimizzando tassi di falsi-positivi. In questo caso, una differente tubazione analisi dovrebbe essere applied elaborare i dati sperimentali e confermare i colpi. </li><li> Seguendo il protocollo per lo screening primario (sezione &quot;Giorno 3: Eseguire Luciferase Assay&quot;), misurare l&#39;attività luciferasi in tutte le piastre CHP134-mycn3UTR e CHP134-CTRL. </li><li> Seguendo il protocollo per lo screening primario (sezione &quot;Giorno 4: Analisi dei dati&quot;), normalizzare i dati sperimentali provenienti da pozzi trattati alla media di sulla piastra comandi del veicolo trattati e calcolare la media e SD su repliche. Per ogni composto testato, calcolare la variazione piega di attività luciferasi in CHP134-mycn3UTR vs. cellule CHP134-CTRL. Selezionare Cambia volte arbitrari cut-off di&gt; 1.5 e significatività p-valori &lt;0,01. NOTA: Nelle impostazioni descritte nel parametro determinante per il successo specificità è un cambiamento significativo di attività volte luciferasi in CHP134-mycn3UTR vs. cellule CHP134-CTRL. Se necessario, si potrebbe anche valutare la tossicità composto eseguendo contemporaneamente un test di vitalità cellulare in seComposti zionato. Questo potrebbe essere particolarmente utile se i colpi principali sono contro-proiettati in un&#39;analisi dose-risposta. Per una singola dose, la nostra esperienza indica una forte correlazione tra l&#39;attività luciferasi ridotta con diminuita vitalità cellulare 6. Pertanto, diminuita attività della luciferasi in cellule bersaglio così come cellule di controllo può essere considerato come un indicatore della tossicità composti, alle concentrazioni testate. </li></ol>

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The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

Per un lungo periodo di regolazione trascrizionale dell&#39;espressione genica è stato pensato per svolgere un importante ruolo, se non esclusivo controllo della produzione di proteine. Accumulando prove, tuttavia, indica che regolazione post-trascrizionale contribuisce tanto quanto, se non di più, regolazione trascrizionale di determinare proteina cellulare abbondanza 1,2. Controllo post-trascrizionale dell&#39;espressione genica è molto più complesso ed elaborato di quanto non fosse in primo pensiero. In realtà, tutte le varie fasi di controllo post-trascrizionale sono emerse da disciplinare, tra cui l&#39;elaborazione mRNA, localizzazione, fatturato, traduzione 3, nonché il nuovo descritto reversibile RNA metilazione 4. Da una serie di processi di regolazione post-trascrizionali potenzialmente interessati, il saggio di seguito descritto si concentra su quelli che coinvolgono &#39;regione non tradotta (3&#39; del mRNA 3 UTR). 3 &#39;UTR colpisce ampiamente mRNA destino principalmente attraverso specifica interazione tra RNA-binding proteins e RNA non codificanti per le sue sequenze di regolazione e / o strutture secondarie 5. Perciò, piccole molecole che modificano queste interazioni o interferiscono con a monte trasduzione del segnale sarà spostare l&#39;equilibrio che regola la proteina abbondanza. Questo approccio terapeutico è particolarmente importante per le patologie umani per i quali esistono evidenze che dimostrano che geni malattia rilevanti sono sottoposte a regolazione post-trascrizionale, che rappresenta quindi un potenziale bersaglio per l&#39;intervento farmacologico. Pertanto, i sistemi che consentono per lo screening dei modulatori livelli di mRNA &#39;attraverso l&#39;interferenza con i meccanismi di controllo post-trascrizionale in un formato ad alta velocità possono diventare strumenti utili all&#39;identificazione di potenziali trattamenti innovativi. Il saggio basato su cellule gene reporter descritto consente l&#39;identificazione di composti capaci di modulare mRNA destino attraverso meccanismi dipendenti dalla sua 3 &#39;UTR. Si compone di diverse parti principali componenti (Figura 1) e richiede pochi passi preliminari per convalidare la fattibilità del test. Prima di tutto, il costrutto giornalista è progettato per includere UTR 3 &#39;da un gene di interesse. Il 3 &#39;UTR si fonde alla fine di un gene reporter luciferasi di lucciola come (Figura 1). Eventuali cambiamenti di meccanismi di controllo post-trascrizionali esercitate attraverso l&#39;inserimento 3 &#39;UTR altereranno la stabilità e / o l&#39;efficienza della traduzione di questa trascrizione chimerico, causando diversi livelli luciferasi. Pertanto, le variazioni di attività luciferasica servono come misura indiretta della colpita regolazione post-trascrizionale del gene di interesse. Il secondo componente del sistema è un costrutto di controllo giornalista, un plasmide di espressione identica che esprime lo stesso gene reporter, ma non contiene alcun 3 &#39;UTR. I due plasmidi reporter possono essere progettati in laboratorio utilizzando protocolli di biologia molecolare convenzionali o acquistati da fonti commerciali. La selezione di una linea cellulare appropriata è fondamentale per la costruzione dosaggio. Quale linea cellulare è il modello ottimale dipende da numerosi fattori che vanno dai requisiti clinici come somiglianza massima alla patologia a questioni solo pratici come caratteristiche disponibilità, transfectability, e la crescita. È importante sottolineare che, prima di procedere si deve fare in modo che la fusione del 3 &#39;UTR al gene reporter ha un effetto previsto sul livello di trascrizione chimerico. L&#39;assenza di una significativa differenza di segnale luciferasi dal 3 &#39;UTR-cuscinetto e controllo giornalista costruisce trasfettate nelle celle selezionate indicherebbe che il 3&#39; UTR non è funzionale in questo modello cellulare, spingendo per la ricerca di quelli alternativi. Per il formato ad alta velocità, le 3 &#39;UTR portante e controllo costrutti reporter luciferasi dovrebbero essere stabilmente integrati nella linea cellulare selezionata. Utilizzando un stabilmente integrato su transitoriamente trasfettatelinea cellulare è preferibile per diverse ragioni. Questo amplierà la scelta di un modello di cellulare, incluse le linee di cellule di difficile trasfezione, diminuire la variabilità dei dati derivanti dalle fluttuazioni di efficienza di trasfezione, abbasserà i costi. Inoltre, su cellule trasfezione transiente sono molto spesso sovraccarico di un plasmide di DNA che porta alla saturazione del sistema. In queste impostazioni un ulteriore aumento dell&#39;espressione giornalista potrebbe essere difficile, con conseguente diminuzione della sensibilità verso i potenziali composti upregulating. Infine, quando tutti i componenti del dosaggio sono impostati, è fondamentale prima di passare al formato high-throughput per determinare la fattibilità del test, vale a dire, se l&#39;attività luciferasi può essere davvero up- e down-regolato specificamente tramite inserito 3 &#39; UTR. I migliori controlli sarebbero piccole molecole segnalati per modulare la stabilità o traducibilità dell&#39;mRNA attraverso UTR 3 &#39;di interesse. Se avere questi ènon è possibile, si accettano controlli surrogate imitano l&#39;effetto desiderato. Questi potrebbero essere miRNA o RNA-binding proteins noti per esercitare i loro effetti attraverso il legame al 3 &#39;UTR di interesse. La valutazione di tali controlli prima della proiezione principale indica non solo la funzionalità del test sviluppato, ma consente anche per la stima della sua gamma dinamica, sensibilità e prestazioni nel formato ad alta produttività. Utilizzando il protocollo descritto abbiamo proiettato una biblioteca di 2.000 composti 6. Neuroblastoma, il tumore solido extracranico più comune dell&#39;infanzia, è stato utilizzato come modello biologico e la 3 &#39;UTR dell&#39;oncogene MYCN, la cui amplificazione predice fortemente esito negativo di neuroblastoma, come gene bersaglio 7. Figura 2 ne illustra lo schema sperimentale. La proiezione è stata divisa in tre corse con la dimensione del lotto di 27 lastre proiettati in una corsa. Il lotto di 27 piatti inclusi biblioteca Spectrum CollectionPiastre n.1-n.8 + n.25 (prima esecuzione),-n.16 n.9 + n.25 (seconda manche) e n.16, n.24 + n.25 (terza pista), ciascun Piastra proiettato in triplice copia. Così, una piastra biblioteca (n.25) è stato analizzato in tutti i tre piste che rendono possibile il confronto inter-run. Il protocollo che segue descrive una singola esecuzione di 9 dischi biblioteca testati in triplice copia.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:239px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Culture plate 96-well White</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6005688</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">StorPlate 96-well U bottom (dilution plate)</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6008190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">100 ml disposable trough for reagents</td>
			<td style="width:239px;">Tecan</td>
			<td>10 613 048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">300 ml disposable robotic reservoir</td>
			<td style="width:239px;">VWR</td>
			<td style="width:119px;">PB12001301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 50&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 200&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td align="right">3711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ONE-Glo Luciferase Assay System&#160;</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">E6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">RPMI</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE12-918F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">PBS-1X, w/o Ca++, Mg++</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">L-Glutamine 200mM</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">The Spectrum collection (compound library)</td>
			<td style="width:239px;">MicroSource Discovery Systems</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan Freedom EVO200 robot&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan F200 multiplate reader&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

Sia trascrizionale e regolazione post-trascrizionale hanno un profondo impatto sulla espressione dei geni. Tuttavia, i saggi di screening basati su cellule comunemente adottati concentrarsi sulla regolazione trascrizionale, essendo essenzialmente finalizzata alla identificazione di molecole promotore-targeting. Come risultato, meccanismi post-trascrizionali sono ampiamente privi di espressione genica sviluppo di farmaci mirati. Qui si descrive un saggio basato su cellule finalizzata a studiare il ruolo di &#39;regione non tradotta (3&#39; UTR 3) nella modulazione del destino del suo mRNA, e ad identificare composti in grado di modificarlo. Il saggio si basa sull&#39;impiego di un costrutto reporter di luciferasi contenente la 3 &#39;UTR di un gene di interesse stabilmente integrati in una linea cellulare malattie rilevanti. Il protocollo è diviso in due parti, con la messa a fuoco iniziale sul screening primario finalizzato alla identificazione di molecole che influenzano l&#39;attività della luciferasi dopo 24 ore di trattamento. La seconda parte del protocollodescrive the-counter di screening necessario per discriminare i composti modulanti attività luciferasi in particolare attraverso UTR 3 &#39;. Oltre al protocollo dettagliato e risultati rappresentativi, forniamo importanti considerazioni circa lo sviluppo del test e la validazione del colpo (s) sul bersaglio endogena. Il reporter assay gene cell-based descritto permetterà agli scienziati di identificare molecole che modulano i livelli di proteina attraverso meccanismi post-trascrizionali dipendenti da una &#39;UTR 3.

Utilizzando l&#39;approccio descritto, abbiamo proiettato una biblioteca 2.000 composti per potenziali modulatori di meccanismi di controllo post-trascrizionale esercitata attraverso la 3 &#39;UTR del gene MYCN. La figura 3 illustra i risultati dello screening primario esemplificato da una singola piastra di libreria. Segnale luciferasi visualizzato come percentuale dei controlli trattati con veicolo è stata ottenuta misurando l&#39;attività luciferasica in triplicato in piastre di cellule CH...

Watch this Scientific Journal Video about High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes at JoVE.com

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

high-throughput-screening-for-chemical-modulators-post

Research

JoVE Journal

Biology

9.1K Views.  University of Trento. Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3’ UTR.

Video: High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening e biopercezione con fluorescente C. elegans Ceppi

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Ricerca

Biologia

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Throughput Screening elevati di attività Endoglucanase fungine in Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

High-throughput per l&#39;analisi Saccarificazione materiali lignocellulosici

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un test di screening a fluorescenza per l&#39;identificazione di modulatori Canali GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening per la piccola molecola modulatori di canali di potassio Inward Rectifier

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

High-throughput screening funzionale utilizzando una casalinga Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificazione delle coppie Kinase-substrato Usando High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.