Ringraziamo Giovanni Darga del DNA Nucleo MGH per la preparazione della biblioteca di screening del DNA. Christopher Chigas di Perkin Elmer ha fornito il supporto prezioso per la nostra Wallac 1420 luminometro. Steven Ciacco e Martin Thomae di Agilent fornito il supporto per il robot Bravo. Ringraziamo Ron Johnson e Steve Tito presso il NIH di generosità fornendo il loro protocollo del saggio di luciferasi dual-bagliore.

Per una panoramica sperimentale, vedi Figura 1. 1. Preparare plasmidi reporter contenente la alfa-sinucleina Promoter Elementi regolatori del gene alfa-sinucleina (Figura 2) si estendono circa 10 kb, da monte NACP (non Un componente di beta amiloide) sequenza dinucleotide repeat 1,2 attraverso introne 2 3. Includiamo una porzione di questa regione in il nostro costrutto giornalista. Abbiamo trovato che i plasmidi contenenti introni 2 richieste procedure speciali per la crescita, come indicato di seguito. I plasmidi di luciferasi, pGL4.10 e pGL4.75 (Figura 3), sono disponibili in commercio da Promega e possono essere propagate utilizzando protocolli di biologia molecolare convenzionali. <ol><li> Amplificare i 2 componenti del promotore alfasinucleina in PCR separati utilizzando la ricetta nella Tabella 1 e primer in Tabella 2. </li><li> Verify prodotti di PCR su un gel di agarosio e pulite utilizzando il kit di purificazione PCR Qiaquick (Qiagen), eluendo in 50 ml di TE (Tris-EDTA). Conservare il eluito 0,9 kb frammento a -20 ° C per un uso futuro. </li><li> Digerire l&#39;intero volume del 3,4 kb frammento di PCR, e 5 ug di pGL4.10, con SacI e XhoI. Trattare il vettore con il vitello Intestino fosfatasi alcalina (Roche) e gel purificare utilizzando il kit PureLink rapida Gel Extraction (Invitrogen). </li><li> Legare il frammento e il vettore utilizzando T4 DNA ligasi (NEB). Pulire la reazione compilato utilizzando il kit PureLink PCR Micro (Invitrogen), eluizione in 10 microlitri di buffer TE. </li><li> Trasforma 2 ml di pulito, reazione di ligazione in 20 microlitri di cellule competenti ElectroMAX Stbl4 (Invitrogen) e l&#39;elettroporazione secondo le istruzioni del produttore. Agitare in un incubatore a 30 ° C a 225 rpm per 90 min. Distribuire 2 volumi su piastre LB contenenti 100 mg / ml ampicillina e incubare a 30 ° C per 2 giorni. </li><li> Usando uno stuzzicadenti, selezionare 4-6 piccole colonie per inoculo in 2 ml di TB (Terrific Broth). Coltivare queste culture Miniprep in 30 ° C shaker O / N. </li><li> Purificare il DNA plasmidico da 1,5 ml di ciascuna coltura miniprep utilizzando il kit PureLink HiPure Plasmid Miniprep (Invitrogen). </li><li> Digerire le minipreps con BamHI e elettroforesi su gel di agarosio. Il clone corretto dovrebbe produrre frammenti di 2.8 kb e 4.9 kb. </li><li> Restreak il restante cultura miniprep dalla corretta clone su un LB (Luria Broth) piastra fresca e crescere a 30 ° C per 2 giorni. </li><li> Selezionare una piccola colonia e inoculare una coltura starter da 1,5 ml TB. Agitare O / N a 30 ° C. Non lasciate che la coltura starter crescere fino a saturazione. </li><li> Diluire l&#39;intera cultura di avviamento in 150 ml di TB preriscaldata e agitare a 30 ° C per 2 giorni. Prima di procedere alla fase successiva, con 1,5 ml di questa coltura per un miniprep e digestione supplementare per confermare che il clone non mutano durante replicati<html> on. </li><li> Purificare il DNA plasmidico dal restante coltura utilizzando il kit PureLink HiPure Plasmid Maxiprep (Invitrogen). Rendimenti attesi di questo plasmide intermedio sono 50-150 g per 150 ml di cultura. </li><li> Scongelare il frammento di 0,9 kb congelati nel passaggio 1.2. Ripetere i passaggi 1,3-1,12 per clonare il frammento di 0,9 kb nel plasmide intermedio, digerire invece con KpnI e SacI. Legare, trasformare, selezionare e crescere per maxipreps come sopra. Digestione con BamHI dovrebbe produrre frammenti di 5.8 kb e 2,8 kb. Questo è il plasmide che verrà utilizzato per lo schermo. </li></ol> 2. Preparare Cellule 293T, Reporter, plasmidi e DNA Library for Screening Cellule 293T vengono prima seminate in piastre da 96 pozzetti e trasfettate il giorno seguente. Plasmidi reporter e DNA biblioteca sono diluiti in piastre da 96 pozzetti. Abbiamo ottenuto lastre di trasfezione-grade libreria di DNA dal Nucleo DNA presso il Massachusetts General Hospital (ottenere = &quot;_blank&quot;&gt; dnacore.mgh.harvard.edu). Questo protocollo transfects una singola piastra di libreria in 2 piastre identiche di cellule 293T (Figura 1), quindi 2 piastre di celle deve essere seminate per ciascuna piastra biblioteca da schermare. Nota: Coltivare cellule in media senza rosso fenolo o antibiotici, in quanto questi interferiscono con i saggi di luciferasi e aumentano la tossicità durante la transfezione, rispettivamente. <ol><li> Per ciascuna piastra libreria da proiettato, risospendere le cellule 293T trypsinized ad una concentrazione di 1,5 x 10 5 cellule / ml in DMEM contenente 14% FBS. Versare in un serbatoio sterile (Axygen). </li><li> Posizionare il serbatoio, 2 chiare a fondo, piastre da 96 pozzetti (Corning), e una scatola di suggerimenti filtro pipetta (Agilent) sul ponte robot. Pipettare 100 microlitri di cellule in ciascun pozzetto di entrambe le piastre, per una densità di 1,5 x 10 4 cellule per pozzetto. </li><li> Centrifugare le piastre cellulari a 50 xg per 2 minuti, utilizzando una velocità di rampa basse per garantire la paritàdensità delle cellule durante ciascun pozzetto. Incubare a 37 ° C e 10% di CO 2 O / N. </li><li> Diluire la lucciola e Renilla plasmidi reporter a 5 ng / ml in mezzi privi di siero. Combinare le diluizioni in un rapporto di 05:03 lucciola di Renilla plasmide (in volume) in un tubo da 15 ml. </li><li> Preparare la plasmidi combinati in ciascun pozzetto di una piastra a 96 pozzetti, dosaggio 17 ml per piastra biblioteca, oltre a 2-10 ml in eccesso, in ogni pozzetto. </li><li> Ottenere trasfezione-grade lastre biblioteca DNA come sopra e diluire a 13,3 ng / ml con acqua priva di endotossine. Il volume minimo per bene dovrebbe essere di 28 ml. </li></ol> 3. Eseguire trasfezioni Il giorno 2 dello schermo, plasmidi reporter e DNA libreria sono trasfettati in cellule 293T. <ol><li> Per ogni piatto biblioteca, mescolare 107,5 ml di RT Optifect (Invitrogen) con 4,3 ml di mezzi privi di siero (1:40 diluizione) in una provetta di polistirene. Incubare per 5 min a temperatura ambiente, e lan dispensare 44 microlitri (per piastra biblioteca) in ciascun pozzetto di una piastra a 96 pozzetti, polistirolo V-fondo (Greiner). </li><li> Posizionare la piastra di Optifect diluito, plasmidi reporter (Passo 2.5), DNA biblioteca (Punto 2.6), e una scatola di puntali sul ponte robot. </li><li> Aspirare 17 ml di plasmidi reporter, 43 ml di Optifect diluita, e 26 ml di DNA biblioteca, inserendo una intercapedine d&#39;aria 5 microlitri tra ogni aspirazione. Il DNA biblioteca dovrebbe essere aspirato ultimo per evitare la contaminazione incrociata. Versare il contenuto delle punte in una nuova lastra di polistirene V-fondo, mescolare, coprire e mettere da parte per 20 minuti per consentire complessi lipide / DNA per formare. Se trasfettando piatti biblioteca multipli, sostituire i puntali e la piastra biblioteca, e ripetere. </li><li> Scoprire la piastra miscela di Optifect / DNA e posizionarlo sul ponte robot con 2 piastre di cellule 293T. Utilizzando un unico box di puntali, aspirare 80 ml di Optifect: miscela di DNA e lentamente dispensare 40 microlitri su ogni piatto di cellule. Se transfezione piatti biblioteca multiple, ripetere per tutti Optifect: lastre DNA. Coprire le cellule. </li><li> Centrifugare piastre delle celle a 1.200 xg per 30 min. Coprire e tornare alla incubatrice. </li><li> Incubare le cellule per 4-6 ore. Durante questa incubazione, preparare supporti fresco miscelando 12 ml di DMEM contenente 10% FBS e 10 pg / ml ciprofloxacina (Cellgro) per ciascuna piastra biblioteca transfettate. Versare mezzi freschi in un serbatoio sterile. </li><li> Posizionare 2 scatole di punte robot, una singola piastra di cellule, il serbatoio contenente terreno fresco, e un serbatoio di rifiuti sul ponte robot. Aspirare 100 microlitri di media dalle cellule e dispensare nel serbatoio rifiuti parzialmente riempito con etanolo al 70%. Usando le punte fresche, aspirare 100 ml di mezzi freschi e lentamente dispensare sulle celle. Ripetere l&#39;operazione per tutte le piastre di cellule. </li><li> Piastre Ritorno al incubatore per 48 ore. </li></ol> 4. Eseguire Dual-luciferasi saggi Il giorno 4 dello schermo, lalucciola e Renilla saggi di luciferasi vengono eseguiti. Nota: Il dosaggio dual-luciferasi richiede l&#39;aggiunta sequenziale di 2 buffer di saggio, 3X tampone lucciola saggio e tampone di saggio 3X Renilla, come descritto nella Tabella 3 di lucciola e Renilla luciferasi non sono secreti, così le cellule devono essere lisate prima di misurare l&#39;attività di luciferasi. . Tampone di saggio lucciola lisa le cellule e fornisce substrato per la luciferasi di lucciola; tampone Renilla estingue il segnale lucciola e fornisce substrato per la luciferasi Renilla. <ol><li> Per ciascuna piastra biblioteca, preparare 8 ml di tampone 3X lucciola saggio da soluzioni madre come descritto nella Tabella 3, e dispensare 82 microlitri in ciascun pozzetto di una piastra a 96 pozzetti con fondo a V. </li><li> Per ciascuna piastra biblioteca, inoltre preparare 12 ml di tampone 3X Renilla e dispensare 122 microlitri in ciascun pozzetto di una piastra a 96 pozzetti con fondo a V. Mettere da parte sia l&#39;lucciola e Renilla buffer di analisi. </li><li> Posizionare una casella di puntali, un serbatoio di rifiuti, e 2 piatti di cellule (trasfettate dallo stesso piatto biblioteca) sul ponte robot. </li><li> Aspirare 60 ml di mezzi di comunicazione di ogni piatto e gettare nel serbatoio rifiuti parzialmente riempito con il 70% di etanolo. Piastre di copertura e mettere da parte. Se trasfettando piatti biblioteca multipli, ripetere per tutti i set di piatti. </li><li> Mettere 2 scatole di puntali, il piatto di 3X tampone di lucciola saggio, e una serie di piatti cellulari sul ponte robot. </li><li> Aspirare 40 ml di 3X tampone di lucciola saggio e aggiungere al primo piatto di cellule, mescolando bene. Il passaggio a nuovi puntali, ripetere per la seconda piastra di cella. Posizionare cellule a temperatura ambiente per 10 minuti per completare la lisi. Se trasfettando piatti biblioteca più, sostituire le scatole di punte e piastre cellulari, e ripetere. </li><li> Record luminescenza da ciascun pozzetto di ciascuna piastra di cella su un luminometro, registrando per 1 sec per pozzetto. Piastre Ritorno al robotdeck. </li><li> Mettere 2 scatole di puntali, il piatto di tampone 3X Renilla, e una serie di piatti cellulari sul ponte robot. </li><li> Aspirare 60 ml di tampone 3X Renilla, e aggiungerlo al primo piatto di cellule, mescolando bene. Il passaggio a una nuova scatola di puntali, ripetere per la seconda piastra di cella. Se trasfettando piatti biblioteca multipli, ripetere per tutti i set di piatti cellulari. </li><li> Record luminescenza da tutte le piastre su un luminometro, registrando ogni pozzetto per 1 sec come sopra. Eliminare le piastre. </li></ol> 5. Analisi dei dati <ol><li> Calcolare la lucciola: rapporto Renilla luminescenza (&quot;F: rapporto R&quot;) per ogni pozzetto dividendo conti di segnale lucciola dai conti di segnale Renilla. </li><li> Calcolare il valore di induzione dividendo il F: rapporto R di ciascun pozzetto dalla F media: rapporto R della piastra, escludendo il più alto e più basso del 25% dei pozzi. </li><li> La media dei valori di induzione di tutti replicatiper una piastra biblioteca transfettate. I geni che inducono o reprimendo l&#39;alfa-sinucleina più di tre pieghe sono considerati &quot;hits&quot; in questa schermata e sono soggetti a ulteriore convalida e schermi secondari. </li></ol>

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Questo lavoro è stato sostenuto da royalties ottenute con BacMamreagents licenze (US Patent 5.731.182).

Alfasinucleina è stato implicato nel morbo di Parkinson (PD) come componente di corpi di Lewy 4, inclusioni intracellulari considerati patognomonici per la malattia. Numerosi studi di associazione genome-wide hanno collegato polimorfismi a singolo nucleotide in alfa-sinucleina con aumentato rischio di sporadici PD 5,6,7. Sebbene meno comune rispetto sporadici PD, PD familiare può essere causato anche da mutazioni nel alfa-sinucleina 8, così come la duplicazione e triplicazione della a...

La capacità di identificare i principali elementi regolatori genetici ed i fattori che agiscono su di essi è fondamentale per l&#39;esplorazione di numerosi processi biologici. Tuttavia, identificando i fattori che regolano l&#39;espressione dei geni in tipi di cellule rare, come specifiche popolazioni di neuroni, può essere impegnativo. Qui vi presentiamo un protocollo per identificare nuovi regolatori trascrizionali di alfa-sinucleina (SNCA), un gene associato con la malattia di Parkinson ed espresso nei neuroni dopaminergici nella substantianigra pars compacta regione del mesencefalo. Compiamo questo utilizzando uno schermo giornalista high-throughput, dual-luciferasi, in vitro decostruire l&#39;espressione di alfa-sinucleina in cellule 293T. Il promotore alfasinucleina viene clonato in un plasmide contenente il gene reporter di luciferasi di lucciola. Un plasmide disponibile in commercio, contenente la luciferasi Renilla sotto il controllo di un promotore costitutivamente attivo serve come controllo interno. Thescostrutti e giornalista sono co-trasfettati in cellule 293T in micropiastre con plasmidi da una libreria di espressione del DNA, in modo che ogni bene è trasfettate con una singola libreria plasmide. Dopo 48 ore, l&#39;attività della luciferasi per ogni giornalista è misurata in sequenza usando un test a doppio bagliore. L&#39;espressione relativa di alfa-sinucleina in risposta a ciascun gene libreria viene dedotto dal rapporto di lucciola: attività di luciferasi Renilla in ciascun pozzetto (F: rapporto R) dopo normalizzazione basato piastra. Questo protocollo consente di schermare un gran numero di geni (~ 2,500 a settimana) per la loro capacità di transattivare un gene reporter utilizzando manodopera minima (1-2 persone) e un costo minimo (circa $ 3 costo reagente per test micropiastra). Regolatori trascrizionali di geni neuronali (ad esempio, alfa-sinucleina), che sono difficili da studiare in neuroni in coltura refrattari alla manipolazione genetica, possono essere confrontati side-by-side in questo saggio funzionale diretta. Noi includiamo nel nostroprotocollo un metodo dettagliato per la clonazione e la crescita dei plasmidi contenenti il ​​promotore alfa-sinucleina, poiché abbiamo scoperto che i plasmidi contenenti questa regione sono instabile quando cresciuta con procedure convenzionali (vedi Discussione). Abbiamo anche un&#39;alternativa economica per i reagenti disponibili in commercio dual-luciferasi per saggi high-throughput. Questo protocollo può essere facilmente adattato per indirizzare altri elementi regolatori di interesse, o su qualsiasi processo che richiede alto throughput trasfezioni transitorie.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput functional screening using a homemade dual-glow luciferase assay

Vi presentiamo un protocollo di screening high-throughput rapido e poco costoso per identificare i regolatori trascrizionali di alfa-sinucleina, un gene associato alla malattia di Parkinson. Cellule 293T sono transitoriamente trasfettate con plasmidi di una libreria di espressione ORF schierati, insieme con plasmidi reporter luciferasi, in un formato micropiastra un gene-per-bene. Attività di luciferasi di lucciola è analizzato dopo 48 ore per determinare gli effetti di ogni gene biblioteca upon alfasinucleina trascrizione, normalizzata a espressione da un costrutto di controllo interno (promotore hCMV dirigere Renilla luciferasi). Questo protocollo è facilitato da un robot banco racchiuso in un armadio biosicurezza, che esegue la movimentazione liquido asettico in formato a 96 pozzetti. Il nostro protocollo di trasfezione automatizzato è facilmente adattabile a high-throughput produzione biblioteca lentivirali o altri protocolli di screening funzionali che richiedono triple-transfezioni di un gran numero di plasmidi biblioteca unica nel coniugatonction con un insieme comune di plasmidi helper. Presentiamo anche un&#39;alternativa economica e validato per disponibili in commercio, doppio reagenti luciferasi che impiega PTC124, EDTA, e pirofosfato di sopprimere l&#39;attività luciferasi di lucciola prima della misura di Renilla luciferasi. L&#39;utilizzo di questi metodi, abbiamo proiettato 7670 geni umani e identificato 68 regolatori di alfa-sinucleina. Questo protocollo è facilmente modificabile per indirizzare altri geni di interesse.

Valori tipici luciferasi, F: tassi di R e valori di induzione per un singolo semipiastra sono mostrati in Figura 4 Nota il colpo in ben H2, un induttore 32 volte.. Geni che causano eccessiva tossicità (per esempio, ben E3), o pozzi che erano scarsamente transfettate, produrranno valori bassi per entrambi lucciola e Renilla luciferasi, ma un valore medio di induzione. Geni che causano induzione non specifico di attività di luciferasi, forse attraverso l&#39;interazione con la spina do...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

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

Vi presentiamo un metodo di screening rapido e poco costoso per identificare regolatori trascrizionali utilizzando high-throughput transfezioni robotici e un dual-bagliore saggio di luciferasi in casa. Questo protocollo genera rapidamente dati scalo side-by-side funzionali per migliaia di geni ed è facilmente modificabile per indirizzare qualsiasi gene di interesse.

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-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.3K Views.  Massachusetts General Hospital.  Vi presentiamo un metodo di screening rapido e poco costoso per identificare regolatori trascrizionali utilizzando high-throughput transfezioni robotici e un dual-bagliore saggio di luciferasi in casa. Questo protocollo genera rapidamente dati scalo side-by-side funzionali per migliaia di geni ed è facilmente modificabile per indirizzare qualsiasi gene di interesse. 

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

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

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 ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manuale Schermo piccole molecole si avvicina ad alta velocità Uso embrioni di zebrafish

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Rivelazione di bersagli miRNA in high-throughput usando il saggio 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

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 ...