SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1. 96 pozzetti lungo Primer PCR <ol><li> Pre-freeze 96 pozzetti PCR più fresco in congelatore -80 ° C. </li><li> In un tubo da 10 ml, preparare Master Mix A: 2.100 ml DDH 2 O, 105 ml PCR grado DMSO, 105 ml 10 mM dNTP, 105 ml modello pPOT (25 ng / ml) 9, per un volume totale di 2.415 ml per piastra . </li><li> Aliquota 23 ml in ciascun pozzetto di una piastra a 96 pozzetti PCR. </li><li> Aggiungere 2 ml di 100 micron pool primer per ogni caricato bene con un P20 12 canali pipetta multicanale. Sigillare con la pellicola, piastra posto sul radiatore PCR pre-raffreddata e permettere di congelare per un minimo di 20 minuti a -80 ° C. </li><li> In un tubo da 10 ml, preparare Master Mix B: 2.048 ml DDH 2 O, 525 microlitri di buffer 10x, 52,5 ml polimerasi per un volume totale di 2.626 ml per piastra. </li><li> Rimuovere la piastra e PCR più fresco dal -80 ° C freezer. </li><li> Con la piastra ancora sul radiatore PCR, aggiungere 25 microlitri Master Mix B in cima alla congelaton Master Mix A per un volume di reazione totale di 50 microlitri. Questo è il tempo critico, e dovrebbe essere completato prima di Mix A può scongelare. Guarnizione della piastra con la pellicola. </li><li> Impostare termociclatore come segue: 94 ° C per 10 min, quindi 30 cicli di 94 ° C per 15 sec, 65 ° C per 30 sec, 72 ° C per 2 min seguita da un periodo di estensione finale di 72 ° C per 7 min . </li><li> Caricare la piastra PCR dopo il blocco ha raggiunto 94 ° C. </li></ol> 2. Convalida dei 96 pozzetti lungo Primer PCR <ol><li> Cast un&#39;ampia 1% (w / v) di gel di agarosio in Tris acetato EDTA (TAE) tampone di corsa con 4 x 28 pozzetti pettini per dare un gel con 4 file di pozzi. Allineare i denti supplenti del pettine con le punte di una pipetta multicanale a 12 canali. immergere con attenzione il gel in TAE tampone di corsa dopo le corsie vengono caricati. </li><li> Carico 1 kb scala DNA nel 1 ° e il 28 ° e di ogni riga. </li><li> Utilizzando un P20 12 canali multicanale pipetta, trasferire 2 ml di PCR dir prodottoectly per ogni corsia di gel (Figura 1A). Fare questo rapidamente per ridurre la possibilità di contaminare gli ampliconi. <ol><li> Caricare i prodotti di PCR di riga A della piastra PCR nelle corsie numerate dispari della prima fila del gel (A1 - corsia 3, A2 - corsia 5 ...... A11 - corsia 23, A12 - corsia 25) e caricare i prodotti di PCR da riga B della piastra PCR nei vicoli numerazione pari del 1 ° fila del gel (B1 - corsia 4, A2 - corsia 6 ...... B11 - corsia 24, B12 - corsia 26). </li><li> Caricare i prodotti di PCR di fila C e D della piastra PCR utilizzando lo stesso modello come descritto sopra, con fila C in corsie numerate e le fila D dispari nelle corsie pari. Continuare questo modello loading finché ogni pozzetto della piastra PCR viene caricato sul gel. buffer di DNA di caricamento non è tenuto a caricare questo gel. </li></ol></li><li> Posizionare il gel nel serbatoio esecuzione e riempire il serbatoio con TAE tampone di corsa fino a quando il gel viene sommerso. Eseguire a 100 V per 30 minutivisualizzare utilizzando un transilluminatore UV. Vedi Figura 1B per un gel esempio. Nota: PCR prodotti possono essere conservati a -20 ° C per diverse settimane prima trasfezione </li></ol> 3. Trasfezione 96 pozzetti <ol><li> Utilizzare il T. brucei prociclico linea di cellule forma SMOXP9 10 per questa procedura e crescere in SDM-79 supporti contenenti il 10% di FCS 10. </li><li> Utilizzare 1 x 10 7 cellule per trasfezione (totale di 1,1 x 10 9 cellule) quando codifica sul terminale N della proteina e 2 x 10 7 cellule per trasfezione (totale di 2,2 x 10 9 cellule) quando codifica sul terminale C di la proteina. Mantenere le cellule in mid-log (1,2 x 10 6 -1 x 10 7 cellule / ml) per diversi giorni prima della trasfezione e raccogliere le cellule per trasfezione ad una densità di 5-8 x 10 6 cellule / ml. </li><li> Lasciare prodotti di PCR congelati per trasfezione per scongelare a temperatura ambiente. </li><li> Contare le cellule utilizzando un emeocytometer o contatore di cellule automatica. </li><li> Pellet numero di celle in più provette da 50 ml a 800 xg per 10 min. </li><li> Dopo la centrifugazione Gettare il surnatante e risospendere le cellule in 10 ml cytomix modificato (0,8 mm EGTA, 24 mm KCl, 0,15 mm CaCl 2, 10 mM fosfato di potassio tampone pH7.6, 25 mM HEPES-KOH pH7.6, 2,6 mm MgCl 2, 0.5% (w / v) di glucosio, 100 ug / ml BSA, 1 mM ipoxantina, 144 mM saccarosio) per provetta. Trasferire tutte le soluzioni cellulari ad un singolo tubo e far girare nuovamente a 800 xg per 10 min. </li><li> Dopo centrifugazione, scartare il surnatante e risospendere le cellule in 23 ml di cytomix modificato per una concentrazione finale di 5 x 10 7 cellule / ml. </li><li> Mentre le cellule sono filatura, aggiungere 1 ml di SDM-79 media per ciascun pozzetto di piastre di coltura tissutale 4 x 24 pozzetti. Etichettare il piastre dC e disegnare un anello attorno pozzetto A1 su ogni piatto in pennarello indelebile per aiutare l&#39;orientamento piatto. </li><li> Collegare il gestore piastra al electroporator. Sulla electroporaUnità tor impostare la tensione a 1500 V, la lunghezza dell&#39;impulso di 100 microsec e il numero di impulsi a 12 e l&#39;intervallo di impulso per 500 msec. Sul conduttore piatto, impostare il conteggio degli impulsi a 1. Questo assicura che tale electroporator applicherà un singolo impulso a ciascuna delle colonne 12 della piastra elettroporazione. </li><li> Utilizzando un P200 a 12 canali multicanale pipetta, trasferire le reazioni di PCR dalla piastra PCR per le piastre di elettroporazione 96 pozzetti usa e getta, Gap 4 mm. </li><li> Quando le cellule sono state filate e sono risospesi alla concentrazione finale di 5 x 10 7 cellule / ml (Passo 3.7), trasferire ad un serbatoio di reagenti. Pipettare 200 ml di soluzione di cellule in ciascun pozzetto della piastra elettroporazione utilizzando un P200 a 12 canali pipetta multicanale, mescolare con il prodotto PCR pipettando. </li><li> Usando tessuto rimuovere eventuali goccioline dalla parte superiore della piastra per evitare cortocircuiti. </li><li> Applicare la pellicola sigillante fornito nella confezione piastra alla sommità della piastra elettroporazione, positioning in modo da lasciare i fori per gli elettrodi scoperti nella parte superiore e inferiore di ogni colonna. Evitare di coprire i punti sollevati utilizzati per guidare il film. </li><li> Caricare la piastra di elettroporazione nel gestore piastra e chiudere il coperchio. Premere &#39;Pulse&#39; sull&#39;unità electroporator. </li><li> Dopo l&#39;elettroporazione, trasferire rapidamente le cellule dalla piastra elettroporazione 96 pozzetti alle piastre di coltura tissutale 4 x 24 pozzetti. Per trasferire le cellule, usare un P200 a 12 canali multicanale pipetta con ogni altro punta mancante in modo tale che i restanti 6 punte allineano con i 6 file su un 24-pozzetti di coltura tissutale. Trasferire le cellule elettroporate nei electroporations piastre a 96 pozzetti nel modello predefinito per le piastre di coltura tissutale 24 pozzetti (Figura 2A). </li><li> Preparare le punte per pipette P200 - per ogni 96 pozzetti trasfezione preparare 2 scatole di 96 punte con ogni altra colonna rimosso. <ol><li> pozzi di trasferimento A1 / A3 / A5 / A7 / A9 / A11 sulla piastra elettroporazione 96 pozzetti apiatto Una riga A delle piastre di coltura di tessuto da 24 pozzetti, B1 / B3 / B5 / B7 / B9 / B11 in riga B, C1 / C3 / C5 / C7 / C9 / C11 in riga C e D1 / D3 / D5 / D7 / D9 / D11 in fila D. Dopo il trasferimento di ogni serie di 6 pozzi dalla piastra a 96 pozzetti per la 24-pozzetti, i pozzi della piastra a 96 pozzetti vengono poi lavate con supporti da rispettivi pozzetti in 24- pozzetti e il lavaggio viene poi trasferito di nuovo ai pozzetti nella piastra 24 pozzetti. </li><li> Trasferimento pozzi E1 / E3 / E5 / E7 / E9 / E11 sul piatto elettroporazione 96 pozzetti a 24 pozzetti B riga A, F1 / F3 / F5 / F7 / F9 / F11 in riga B, G1 / G3 / G5 / G7 / G9 / G11 in riga C e H1 / H3 / H5 / H7 / H9 / H11 in fila D. Dopo il trasferimento di ogni serie di 6 pozzi dalla piastra a 96 pozzetti per la 24-pozzetti, i pozzi in 96 piastra -well vengono poi lavati con supporti da rispettivi pozzetti nella piastra 24 pozzetti ed il lavaggio viene poi trasferito nuovamente ai pozzetti nella piastra 24 pozzetti. </li><li> pozzi di trasferimento A2 / A4 / A6 / A8 / A10 / A12 sulla piastra elettroporazione 96 pozzetti a 24 pozzetti C riga A, B2 / B4 / B6 / B8 / B10 / B12 in riga B, C2 / C4 / C6 / C8 / C10 / C12 in riga C e D2 / D4 / D6 / D8 / D10 / D12 in fila D. Dopo il trasferimento di ogni serie di 6 pozzi dal 96 piastra -well alla piastra 24 pozzetti, i pozzi della piastra a 96 pozzetti vengono poi lavati con supporti da rispettivi pozzetti nella piastra 24 pozzetti ed il lavaggio viene poi trasferito nuovamente ai pozzetti nella piastra 24 pozzetti. </li><li> pozzi di trasferimento E2 / E4 / E6 / E8 / E10 / E12 sul piatto elettroporazione 96 pozzetti a 24 pozzetti D riga A, F2 / F4 / F6 / F8 / F10 / F12 in riga B, G2 / G4 / G6 / G8 / G10 / G12 in riga C e H2 / H4 / H6 / H8 / H10 / H12 in fila D. Dopo il trasferimento di ogni serie di 6 pozzi dalla piastra a 96 pozzetti per la 24-pozzetti, i pozzi del 96 piastra -well vengono poi lavati con supporti da rispettivi pozzetti nella piastra 24 pozzetti ed il lavaggio viene poi trasferito nuovamente ai pozzetti nella piastra 24 pozzetti. </li></ol></li><li> Utilizzando un microscopio a contrasto di fase invertito, esaminare 1 e da ogni colonna per controllare le cellule. Ci sarà una miscela di cellule vive e morte connotevole quantità di detriti cellulari. cellule vive possono essere distinte dalle cellule morte perché saranno luminoso al microscopio a contrasto di fase e saranno in movimento. </li><li> Posizionare le piastre da 24 pozzetti in una scatola pulita, di plastica con un coperchio che forma una tenuta ermetica. Mettete la scatola in un incubatore a 28 °. </li><li> Tra 6-8 ore più tardi, aggiungere 1 ml di SDM-79 con 10% FCS contenente antibiotico selettivo 2x a ciascun pozzetto. Nella nostra esperienza, le linee cellulari in cui i geni sono targhetta su terminale N sono resistenti alle alte concentrazioni di farmaco selettivo. Pertanto, utilizzando blasticidin come esempio, codifica sul terminale N richiede una concentrazione finale di 20 ug / ml blasticidin e codifica il terminale C richiede una concentrazione finale di 10 ug / ml blasticidin. Nota: linee cellulari transgeniche saranno visibili 9-10 giorni dopo la trasfezione. </li><li> Passaggio almeno due volte nel farmaco fresco e supporti prima dell&#39;analisi. La Figura 2B mostra i risultati di un tipico Transfe 96 pozzettiction per la codifica di 96 diverse proteine ​​sul terminale N. Valutare ogni bene al microscopio a contrasto di fase per determinare se contiene cellule sane, resistenti ai farmaci. A ben sano contiene prevalentemente (&lt;95%), le cellule altamente attivi che sono luminose nel campo della microscopia contratto di fase. L&#39;analisi successiva mediante microscopia in epifluorescenza o Western Blot è necessaria per determinare se la proteina è stato etichettato con successo. </li></ol>

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The authors have nothing to disclose.

notevoli miglioramenti nella sensibilità di proteomica e trascrittomica metodi negli ultimi 5-10 anni ha fornito dati preziosi su migliaia di geni e dei loro prodotti. Tuttavia, gli strumenti per affrontare la funzione di queste proteine ​​non hanno tenuto il passo. Tagging una proteina facilita numerosi esperimenti per determinare la sua funzione. Ad esempio, una proteina può essere fuso ad una proteina fluorescente in una varietà di colori diversi per facilitare gli studi di localizzazione e co-localizzazione. Tag sviluppato per la microscopia elettronica, come ad esempio APEX2 o miniSOG 11,12, consentire la localizzazione ultrastrutturale della proteina codificata. Parole chiave per la biochimica, come ad esempio il tag TAP, e ProtC-TEV-Prota (PTP) Indicatore 7,13 permettere la purificazione di complessi associati con la proteina per l&#39;identificazione di partner di legame o in saggi biochimici in vitro. I passi specifici che sono cruciali per il successo del ProtoColo sono: l&#39;incorporazione di DMSO nella PCR Master Mix 1, congelamento della PCR Master Mix 1 prima dell&#39;aggiunta del Master Mix 2, l&#39;uso della polimerasi commerciale in Master Mix 2 e la modifica del buffer cytomix elettroporazione. Nella nostra esperienza, è necessario utilizzare il doppio del numero di celle per C trasfezioni terminale di codifica da per N trasfezioni terminale di codifica per ottenere una percentuale simile di pozzetti positivi. Pertanto, tutti i passaggi devono essere eseguiti come descritto. Questa tecnica è solo probabile che sia successo quando transfecting l&#39;insetto prociclico modulo trypanosome. Forme sangue hanno una bassa efficienza di trasfezione 14, inoltre sono probabilità di morire durante la selezione a causa della tossicità densità-dipendente che non è correlato al farmaco selettivo. Pertanto, il nostro precedente protocollo rappresenta la migliore tecnologia attuale per la codifica di tripanosomi forma sangue 9. E &#39;anche probabile che il transefficienza perfezione varia dipende dalla specifica isolato trypanosome. Questo protocollo è stato ottimizzato utilizzando 927 SMOX forme prociclico - altri ceppi possono richiedere ulteriore ottimizzazione. Le misure che possono aumentare la probabilità di successo includono: aumentando la quantità di PCR amplicone, aumentando il numero di cellule inclusi nella trasfezione. Vi presentiamo un metodo in cui centinaia di proteine ​​possono essere contrassegnati in parallelo. Ciò faciliterà studi su larga scala sulla localizzazione e l&#39;interazione, integrando grandi set di dati esistenti e di fornire informazioni preziose per la comunità. modelli Primer sono disponibili su richiesta e un elenco di plasmidi modelli sono disponibili presso: http://www.sdeanresearch.com/

Trypanosoma brucei è un protozoo parassita che causa trypanosomasis africana umana e nagana nei bovini. T. brucei è un organismo ideale per l&#39;analisi della funzione proteica grazie alla combinazione di un genoma di alta qualità, numerose proteomica e trascrittomica set di dati e strumenti molecolari ben sviluppati 1-3. I progressi nella proteomica e il sequenziamento hanno portato a grandi serie di dati che evidenziano potenzialmente geni interessanti 4-6; Tuttavia, molti geni hanno informazioni minime ad essi associati nei database esistenti. Vi è quindi la necessità di un metodo di alta produttività per aiutare caratterizzazione funzionale della proteina. Espressione di una proteina marcata può dare una molteplicità di intuizioni in funzione di una proteina. Ad esempio, una proteina marcata con una proteina fluorescente o epitopo può essere localizzato mediante microscopia a fluorescenza, che fornisce informazioni su dove protein potrebbe essere esercitando il suo effetto biologico. In alternativa, una proteina contrassegnati con un TAP 7, 8 o HaloTag sua etichetta può essere purificato per saggi biochimici e l&#39;identificazione dei suoi partner di interazione. Recentemente abbiamo sviluppato una metodologia di codifica robusta per T. brucei 9. Questo era lungo Primer PCR per generare il DNA per trasfezione e ha permesso la marcatura di decine di proteine ​​in parallelo - un miglioramento importante di protocolli esistenti. Ora abbiamo migliorato la scalabilità di questo protocollo in forme prociclico da un ordine di grandezza. Qui, vi presentiamo il nostro metodo in cui eseguiamo la PCR e trasfezione in piastre a 96 pozzetti, con il recupero e la selezione che si verificano in piastre da 24 pozzetti. Come centinaia di proteine ​​possono essere contrassegnati in parallelo questo metodo fornisce un metodo conveniente e fattibile per la codifica l&#39;intero genoma trypanosome.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotide</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:119px;">na</td>
			<td style="width:167px;">desalt only (do not use additional purification)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMSO</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">Included with the Expand HiFi pack</td>
			<td>Must be PCR grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP</td>
			<td style="width:115px;">any reputable</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand HiFi polymerase</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">11759078001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTX ECM830</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td>Electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HT-200 plate handler</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">HT-200</td>
			<td>Handles the 96-well eletroplates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOS 96 ELECTROPLATE 4mm</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">45-0452</td>
			<td>Electroplate for the 96-well electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Melford</td>
			<td style="width:119px;">3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin b Gold</td>
			<td>Invivogen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phleomycin</td>
			<td style="width:115px;">Melford</td>
			<td style="width:119px;">P0187</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">225cm TC Flask, canted neck, phenolic cap</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well culture plates</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC017</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eppendorf&#174; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well Multiply&#174; PCR plate with lateral skirt</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:119px;">72.1979.203</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput gene tagging in trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

In questo trasfezione rappresentante, i primer sono stati progettati utilizzando lo script perl Tagit 9 e sintetizzati in commercio. Il 96 pozzetti PCR è stato eseguito e convalidato come descritto (Figura 1A); in questo esempio, 95/96 PCR hanno avuto successo (Figura 1B). Nella nostra esperienza, ripetendo le reazioni fallite sia con i primer originali o primer ri-sintetizzato non si traduca in una PCR di successo. Gli ampliconi sono state trasferite in piastre da 96 pozzetti elettroporazione per elettroporazione (Figura 2A) e poi trasferiti piastre di coltura da 24 pozzetti per il recupero e la selezione. Dopo un tempo sufficiente a permettere la selezione avvenga, i pozzetti sono stati ottenuti per la sopravvivenza prima ulteriori analisi. In questo esempio, 88/96 pozzi (92%) sono risultati positivi dopo la selezione di 15 giorni. <img alt = &quot;Figura 1&quot; src = &quot;/ files / ftp_upload / 54342 / 54342fig1.jpg&quot; /&gt; Figura 1: convalida di lunga Primer PCR (A) del modello per il trasferimento di prodotti di PCR da 96 pozzetti PCR per il gel per la validazione della PCR.. (B) A 96 pozzetti PCR gel rappresentante convalida per la codifica 95 geni sul terminale N e 1 amplicon non contenente 5 &#39;omologia di targeting per fungere da controllo negativo nella trasfezione. I campioni vengono caricati su un 1% (w / v) gel e risolti a 100 V per 30 min. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54342/54342fig2.jpg" /> Figura 2: 96 pozzetti elettroporazione e la selezione (A) del modello per il trasferimento di cellule elettroporate da 96 pozzetti elettroporazione plat.e per 24 pozzetti piastre di coltura dei tessuti. (B) Un schematicamente una tipica competenza live / morti dopo 15 giorni di selezione in piastre da 24 pozzetti. Verde rappresenta un trasfezione successo, grigio rappresenta un pozzo con solo cellule morte. In questo esempio, 88/96 (92%) dei pozzi conteneva parassiti transfettate con successo. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput di Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

high-throughput-gene-tagging-in-trypanosoma-brucei

Research

JoVE Journal

Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei

7.9K Views.  University of Oxford. Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Video: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

L&#39;esame dei telomeri G-Struttura in aggetto Trypanosoma brucei</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Ricerca

Immunologia e infezioni

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitativa High-throughput cella singola test di citotossicità per le cellule T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

rilevamento di<em&gt; Trypanosoma brucei</em&gt; Switching Variante superficie glicoproteina da magnetico cellulare Attivato Ordinamento e citometria a flusso

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detection High-throughput respiratorie patogeni nei campioni animali da Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Costruire una piattaforma di High-throughput Screening per valutare l'eterogeneità dell'amplificazione del Gene HER2 nei cancri al seno

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un ad alta velocità, ad alto contenuto, liquido a base di c. elegans patosistema come contributo

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Misurazione di High-throughput di membrana plasmatica risigillazione efficienza in cellule di mammifero

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Alta velocità effettiva In Vitro valutazione della latenza inversione agenti sulla trascrizione di HIV e Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...