Il lavoro è stato sostenuto dalla concessione nr. 01GU0615 del &quot;Bundesministerium für Forschung und Technologie (BMBF).

1) Consegna batterica di shRNAs <ol><li> Prima di iniziare con la coltura batterica, si deve preparare LB-media e LB-agar. </li><li> Per la LB-medio pesare estratto di lievito (0,5% w / v), bacto triptone (1,0% w / v) e NaCl (0,6% w / v) e diluire questi componenti in acqua bidistillata. Le soluzioni preparate devono essere sterilizzati in autoclave e sono pronti per l&#39;uso. </li><li> Per LB-agar agar bacto (1,5% w / v) deve essere aggiunto al LB-medium precedente alla sterilizzazione. </li><li> Riscaldare LB-agar nel forno a microonde fino a quando il LB-agar è completamente sciolto. Lasciar raffreddare la soluzione fino a quando la bottiglia può essere toccato con facilità senza bruciarsi. Aggiungi kanamicina (100 mg / ml) per la selezione di cloni positivi in ​​fasi successive. </li><li> Preparare LB-agar piastre a una panchina a flusso laminare (condizioni sterili) pipettando 20 ml di LB-agar soluzione in 10 cm di Petri-piatti. Lasciate raffreddare le piastre fino a quando il liquido è diventato solido. Le piastre sono ora pronti per l&#39;uso e può essere conservato a 4 ° C. </li><li> ShRNA-encoding vettori di espressione sono trasformati in competenti E. coli ceq221 da shock termico utilizzando la procedura di CaCl 2. </li><li> Piastra i batteri trasformati su LB-agar in piastre contenenti 100 mg / ml kanamicina, chiudere il coperchio, sigillare le piastre con parafilm e coltivare a testa in giù a 37 ° C durante la notte. </li><li> Inoculare batteri culture mini con cloni positivi con la scelta di colonie cresciute con uno stuzzicadenti. Trasferire il stuzzicadenti in 7,0 ml di fresca LB-mezzo contenente 100 mg / ml kanamicina e coltivare a 37 ° C durante la notte su un agitatore a 200 giri al minuto. </li><li> Inoculare 100 ml di fresca LB-mezzo contenente 100 mg / ml kanamicina 1:100 con la cultura notte su mini in un pallone di Erlenmeyer l 1, e incubare a 37 ° C durante la notte su un agitatore a 200 giri al minuto. </li><li> Semi di 25 x 10 4 (numero di cellule inseminate dipende dalla velocità di crescita delle cellule della linea cellulare secondo; confluenza dovrebbe essere il 70-80% ~ 24 ore dopo la semina) cellule umane di carcinoma gastrico (EPG85-257RDB) per bene in 6 - piatti bene e incubare questi a 37 ° C, in atmosfera al 5% di CO 2, satura di vapore acqueo durante la notte. </li><li> Precedente infezione di cellule di cancro, oltre culture notte del tk RNAi vettore contenente E. coli sono misurati in un fotometro per determinare il diametro esterno 600. Diluire la soluzione batterica fino a una densità ottica di 0,5 si raggiunge (600 OD = 0,5 è uguale a 1,6 x 10 8 cellule batteriche / ml). </li><li> Sostituire la FCS-contenente terreno di coltura di cellule del carcinoma a cellule contro FCS-free medio 30 minuti precedenti batterica co-incubazione. </li><li> Lavare due volte i batteri con 1 x PBS, e diluire nel siero senza Leibovitz L-15 di media. </li><li> Aggiungi batteri diluito per le cellule tumorali al desiderato MOI (molteplicità di infezione = numero di cellule batteriche per cella cancro) e co-incubare i batteri e le cellule tumorali per 2 ore a 37 ° C. </li><li> Dopo 2 ore di co-incubazione le cellule tumorali sono state lavate due volte con PBS 1x x e una volta con siero contenente Leibovitz L-15 di media integrato con 100 U / ml di penicillina, 100 mg / ml di streptomicina, 2,5 mg / ml di amfotericina, 150 mcg / ml di gentamicina e 100 mg / ml kanamicina. </li><li> Continuare a coltivare le cellule tumorali a 37 ° C, in atmosfera al 5% di CO 2, satura di vapore acqueo. </li><li> Gli effetti del trattamento batteriche possono essere visualizzati al microscopio a fluorescenza, misurata mediante quantitativa real-time PCR, RT a livello di mRNA, e attraverso l&#39;analisi western blot a livello di proteine, e mostrato con test funzionali come l&#39;analisi FACS, e saggi di proliferazione cellulare. (Se voluto, queste procedure possono essere descritte in dettaglio). </li></ol> 2) Rappresentante Risultati Se il protocollo viene eseguita correttamente, i risultati dovrebbero essere paragonabili a quelli riportati di seguito. Microscopia a fluorescenza La figura 1 mostra una cellula umana carcinoma gastrico tre ore dopo il trattamento batterica (a) in confronto a una cella non trattata (b). Attorno al nucleo della cellula trattata, i batteri possono essere rilevati. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Figura 1" /> Figura 1: la microscopia a fluorescenza di cellule umane di carcinoma gastrico dopo trattamento batterica (1:500) e un trattato di cellule umane di carcinoma gastrico, come controllo, DAPI-colorazione, DAPI filtro passa-banda (Λem = 640 nm), obiettivo 40x. Quantitativa real-time RT PCR Nella figura 2 un regolamento giù di MDR1 mRNA di circa il 70% (fascio nero) possono essere osservati dopo trattamento con i batteri terapeutico. Cellule parentali servire come controllo positivo a causa della mancanza di MDR1 sovraespressione. La linea cellulare 257RDB P170 contenente un plasmide che esprime anti-MDR1 shRNAs s preso come confronto diretto della tecnologia transkingdom RNAi ad altri RNAi tacere strategie. La linea di cellule non trattate resistenti EPG85-257RDB sovraespressione MDR1, la linea stessa cella trattati con batteri privi del shRNA esprimere plasmide, e questa linea di cellule trattate con i batteri terapeutico porta un plasmide che esprime anti-MRP2 shRNAs sono stati presi come controlli positivi. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Figura 2" /> Figura 2: quantitativa real-time PCR, RT espressione di mRNA MDR1 dopo il trattamento terapeutico con E. coli ceq221 esprimere anti-MDR1 shRNAs (MOI 1: 500). La normalizzazione è stata effettuata utilizzando il dell&#39;aldolasi gene housekeeping. Il rapporto MDR1/aldolase di cellule non trattate della linea cellulare EPG85-257RDB sono stati fissati al 100%. P-valori sono stati calcolati utilizzando dello studente t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Western Blot analisi La figura 3 mostra che il MDR1 giù regolamento ha avuto luogo a livello della proteina batterica dopo il trattamento. Essa mostra la mancanza MDR1 espressione del sensibile linea cellulare di droga dei genitori (EPG85-257P), l&#39;espressione di MDR1 non trattata linea cellulare resistente ai farmaci (EPG85-257RDB) e la MDR1 espressione del campione trattato. Un regolamento chiaro giù di MDR1 delle cellule trattate batteri possono essere osservati. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Figura 3" /> Figura 3: analisi di Western Blot MDR1 livelli di espressione dei non trattati farmaco-sensibile EPG85-257P, la non trattati farmaco-resistente EPG-257RDB, e di EPG85-257RDB dopo la co-incubazione con E.. coli ceq221 + P43 MDR1. Ab primario C219 1:100 e anti-actina 1:5 000, secondaria Ab anti-topo 1:10 000. Test di citotossicità Analisi funzionale come il test di citotossicità in figura 4 sono anche indicatori per il funzionamento della RNAi transkingdom. La linea cellulare dei genitori e la linea cellulare contenente un anit-MDR1 shRNA esprimere plasmide non mostrano alcuna resistenza alla daunorubicina. La linea cellulare resistente EPG85-257RDB e due ulteriori controlli non mostrano variazioni significative nella resistenza rispetto al campione trattato con anti-batteri MDR1 shRNA esprimere in cui la resistenza alla daunorubicina potrebbe essere invertita di circa il 90%. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Figura 4" /> Figura 4: test di citotossicità. Farmaco-specifici IC50-valori determinati da un test di citotossicità per la sopravvivenza delle cellule. P-valori sono stati calcolati utilizzando dello studente t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Antracicline accumulo di test Secondo la ridotta resistenza dei campioni batteri trattati (figura 4), l&#39;accumulo delle antracicline anti-cellule MDR1 shRNA trattati è aumentato di circa il 90% come mostrato in figura 5. La linea cellulare dei genitori e la linea cellulare 257RDB P170 mostrano un forte accumulo di daunorubicina fino al 100%. La variante resistente mostra raramente accumulo. Cellule trattate con farmaci anti-batteri MDR1 shRNA esprimere mostrano un aumento anthracyline accumulo del 90%. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Figura 5" /> Figura 5: saggio di accumulazione antraciclina. Accumulo antracicline delle cellule di carcinoma 6 giorni dopo il trattamento batterica misurato mediante citometria di flusso. P-valori sono stati calcolati utilizzando dello studente t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001).

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Il numero di cellule seminate per le infezioni e il MOI corrispondenti usati, dipendono criticamente sulle linee cellulari sotto osservazione e la loro velocità di crescita. Per trovare il numero di cellule ottimale per la semina, pre-esperimenti per determinare la velocità di crescita sono fortemente raccomandati. Oltre a questo, MOIS diverse dovrebbero essere testati a causa della limitata estendere a cui le cellule possono sopportare l&#39;invasione batterica senza morire di stress. Il punto ottimale di tempo in cu...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bacterial delivery of rnai effectors: transkingdom rnai

Interferenza dell&#39;RNA (RNAi) rappresenta un meccanismo efficace per alto inibizione specifica di espressione di mRNA. Oltre alla sua potenzialità come strumento di laboratorio potente, il percorso RNAi sembra essere promettente per l&#39;utilizzo terapeutico. Per lo sviluppo di RNA interference (RNAi) a base di terapie, la consegna di RNAi-mediare agli agenti di cellule bersaglio è uno dei principali ostacoli. Una nuova strategia per superare questo ostacolo è transkingdom RNAi (tk RNAi). Questa tecnologia utilizza batteri non patogeni, ad esempio, Escherichia coli, a produrre e consegnare terapeutici a breve hairpin RNA (shRNA) all&#39;interno delle cellule bersaglio per indurre RNAi. Una prima generazione tk RNAi-mediare vettore, TRIP, contiene il promotore del batteriofago T7 per la regolazione della espressione di un shRNA terapeutiche di interesse. Inoltre, TRIP è il luogo Inv. da pseudotuberculosis Yersinia che codifica invasin, che permette di batteri naturali non invasivi per entrare β1-integrina-positivi cellule di mammifero e il gene HlyA da Listeria monocytogenes, che produce listeriolysin O. Questo enzima permette la shRNA terapeutico per sfuggire ingresso vescicole all&#39;interno del citoplasma della cellula bersaglio. Costruisce TRIP sono introdotti in un competente non patogeni ceppo di Escherichia coli, che codifica per l&#39;RNA polimerasi T7 necessari per il promotore T7-driven sintesi di shRNAs. A ben caratterizzato associata al cancro molecola bersaglio per le diverse strategie di RNAi è ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Questo-trasportatore ABC agisce come una pompa di estrusione di droga e la media &quot;classici&quot; ABCB1-mediata multiresistenza ai farmaci (MDR) fenotipo delle cellule tumorali umane che si caratterizza per un modello specifico resistenza crociata. Diverse ABCB1 che esprimono le cellule tumorali MDR sono stati trattati con anti-ABCB1 espressione vettore shRNA cuscinetto E. coli. Questa procedura ha determinato l&#39;attivazione dei percorsi RNAi all&#39;interno delle cellule tumorali e un regolamento notevole giù del mRNA ABCB1 codifica e la corrispondente pompa di estrusione droga. Di conseguenza, l&#39;accumulazione della droga è stato migliorato nella incontaminata farmaco-resistenti le cellule tumorali e il fenotipo MDR è stata invertita. Per mezzo di questo modello i dati forniscono la proof-of-concept che tk RNAi è adatto per la modulazione di cancro associati fattori, ad esempio ABCB1, nelle cellule tumorali umane.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Per lo sviluppo di RNA interference (RNAi) a base di terapie, una nuova strategia è stata sviluppata, transkingdom RNAi (tkRNAi). Questa tecnologia utilizza batteri non patogeni per produrre e consegnare terapeutici a breve hairpin RNA (shRNA) all&#39;interno delle cellule bersaglio. Qui, tkRNAi stato applicato con successo per l&#39;inversione del classico ABCB1-mediata multiresistenza ai farmaci (MDR) delle cellule tumorali.

Consegna batterica di RNAi per DJ: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

bacterial-delivery-of-rnai-effectors-transkingdom-rnai

Research

JoVE Journal

Biology

13.4K Views.  Charité Campus Mitte. Per lo sviluppo di RNA interference (RNAi) a base di terapie, una nuova strategia è stata sviluppata, transkingdom RNAi (tkRNAi). Questa tecnologia utilizza batteri non patogeni per produrre e consegnare terapeutici a breve hairpin RNA (shRNA) all'interno delle cellule bersaglio. Qui, tkRNAi stato applicato con successo per l'inversione del classico ABCB1-mediata multiresistenza ai farmaci (MDR) delle cellule tumorali.

Video: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocollo per saggi RNAi in zanzare adulte (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Ricerca

Biologia

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identificazione dei fenotipi inibizione della crescita indotta da Espressione di effettori batterica di tipo III nel lievito

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSIONE per lo screening RNAi in cellule di mammifero

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

L&#39;interferenza di RNAi per iniezione in dsRNA Drosophila Embrioni

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi mediata Knockdown Gene e Transgenesi dalla microiniezione nel nematode Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative e automatica High-throughput Genome-wide Schermi RNAi in C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-mediato Knockdown Gene e<em&gt; In Vivo</em&gt; Saggio La diuresi in femmina adulta<em&gt; Aedes aegypti</em&gt; Le zanzare

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Analisi fosfopeptide di roditori Epididymal spermatozoi

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Consegna batterica di RNAi per DJ: Transkingdom RNAi

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Analisi fosfopeptide di roditori Epididymal spermatozoi