Questo studio è stato sostenuto da sovvenzioni da Taiwan (Ministero della scienza e della tecnologia, più 100-2923-B-002-002-MY3 e 106-2313-B-002-011-MY3 a H.J.L.), Repubblica Ceca (concedere Università Agenzia della Boemia meridionale, GAJU concedere 065/2017/P a Y.H.L) e Spagna ( Ministero spagnolo dell'economia e della competitività, concede CGL2012-36251 e CGL2015-64727-P a X.B. e il governo catalano, concedere 2014 SGR 619 a X.B.); ha inoltre ricevuto il sostegno finanziario del Fondo europeo economica e lo sviluppo regionale (fondi FEDER a X.B.).

1. sintesi e preparazione di dsRNA
<ol><li>Identificare i siti di destinazione di dsRNA nella regione 3' non tradotta dei geni bersaglio. La dsTub è utilizzato per il targeting il gene α-tubulina (tub) (numero di accessione GenBank: KX228233), e dsEGFP come un controllo negativo dsRNA è progettato dalla sequenza di intensificare la proteina fluorescenza verde (EGFP; Numero di accessione GenBank: LC311024).</li>
	<li>Eseguire l'amplificazione di PCR standard per sintetizzare i modelli di dsRNA con gli iniettori di gene-specific contenente la sequenza del promotore T7 (5'-TAATACGACTCACTATAGGG-3'). Le condizioni di amplificazione di PCR e informazioni sul primer utilizzati sono quelli segnalati da Lin et al. (2017) 12 (Vedi Tabella materiali).</li>
	<li>Procedere con la preparazione di dsRNA utilizzando una trascrizione di T7 Kit (Vedi Tabella materiali), seguendo le istruzioni del produttore. Nota: Resa elevata quantità dsRNA, mescolare due reazioni in un tubo (in totale 40 µ l) e incubare a 37 ° C durante la notte.</li>
	<li>Aggiungere 2 µ l di dnasi (Vedi Tabella materiali) in campioni di dsRNA per 15 min a 37 ° C per modelli del DNA del digest.</li>
	<li>Aggiungere 200 µ l di reagente di estrazione (Vedi Tabella materiali) e cloroformio 40 µ l, poi vortice.</li>
	<li>Centrifugare per 10 min a 13.000 x g a 4 ° C, quindi raccogliere il surnatante (150 µ l) in un nuovo tubo del microcentrifuge.</li>
	<li>Precipitare dsRNA aggiungendo 150 µ l isopropanolo e incubare per 15 minuti in ghiaccio.</li>
	<li>Centrifugare per 15 min a 13.000 x g a 4 ° C, quindi rimuovere il surnatante.</li>
	<li>Aggiungere 200 µ l di etanolo 70% per lavare dsRNA pellet. Centrifugare per 10 min a 13.000 x g a 4 ° C.</li>
	<li>Rimuovere l'etanolo e ripetere le fasi di lavaggio di etanolo.</li>
	<li>Rimuovere l'etanolo e asciutto dsRNA pellet da concentratori centrifughi vuoto per 3 min, quindi risospendere dsRNA con acqua RNAsi-libera.</li>
	<li>Determinare la quantità del dsRNA purificato utilizzando uno spettrofotometro UV-Vis di micro-volume (Vedi Tabella materiali) secondo le istruzioni del produttore e regolare fino alla concentrazione desiderata (ad es., 0,25 µ g / µ l per la preparazione di dsRNA lipoplessi).</li>
	<li>Conservare i campioni di dsRNA a-20 ° C fino a 3 mesi.</li>
</ol>2. preparazione del dsRNA lipoplessi
<ol><li>Diluire 4 µ l di reagente di transfezione (Vedi Tabella materiali) aggiungendo 4 µ l di soluzione glucosata al 5%. Diluire 4 µ l di dsRNA (1 µ g) aggiungendo 4 µ l di soluzione glucosata al 5%.</li>
	<li>Aggiungere la soluzione di reagente liposoma diluito immediatamente nella soluzione diluita dsRNA tutti in una volta. Vortice brevemente per mescolarli, quindi incubare per 15 minuti a 25 ° C. Nota: Non mescolare le soluzioni nell'ordine inverso.</li>
	<li>I dsRNA lipoplessi sono pronti all'uso. Usare entro 1 ora di preparazione e scartare dopo 1 ora. Nota: Secondo le istruzioni del produttore, il lipoplessi dovrebbero essere usato appena possibile.</li>
</ol>3. raccolta di enzimi extracellulari dall'emolinfa e succo del Midgut
<ol><li>
Insetto buffer saline (stock x 10)
	<ol>
<li>Mescolare 900 mL acqua distillata doppia con 109,3 g NaCl, 15,7 g KCl, 6,3 g CaCl2e 8,3 g MgCl2·6H2O.</li>
		<li>Aggiustare il pH a 6,8 e riempire fino a 1.000 mL.</li>
	</ol>
</li>
	<li>
Collezione di emolinfa
	<ol>
<li>Anestetizzare gli scarafaggi sul ghiaccio fino a quando non si muovono per 3 min.</li>
		<li>Tenere lo scarafaggio con due dita (pollice e indice) e alzare il lato ventrale del scarafaggio.</li>
		<li>Utilizzare le forbici per dissezione per tagliare fuori la punta di una coxa di una gamba posteriore. Nota: Tagliare l'intersezione collegato tra la coxa e il trocantere. L'asportazione della parte del coxa era meno efficiente per raccogliere l'emolinfa.</li>
		<li>Spremere l'addome delicatamente con il dito medio e raccogliere l'emolinfa sanguinamento dall'incisione con una micropipetta 10 µ l.</li>
		<li>Piscina l'emolinfa da scarafaggi (5 persone) in un tubo del microcentrifuge. Nota: Mantenere le provette microcentrifuga sul ghiaccio per prevenire melanizzazione. Il volume medio di emolinfa ottenuto da uno scarafaggio maschio è 2-3 µ l.</li>
		<li>Rotazione verso il basso l'emolinfa brevemente con una centrifuga da banco per 10 s.</li>
		<li>Trasferire il volume desiderato di emolinfa (vale a dire, 10 µ l) e mescolare con 50 µ l di tampone salino insetto 1x.</li>
		<li>Centrifugare per 10 minuti a 1.000 x g a 4 ° C a girare giù emociti. Trasferire il surnatante in una provetta pulita microcentrifuga.</li>
		<li>Quantificare la concentrazione di proteine totali utilizzando uno spettrofotometro UV-Vis di micro-volume misurando A28015.</li>
		<li>Regolare ogni campione per avere la stessa concentrazione di proteina (6 mg / µ l di proteina totale).</li>
	</ol>
</li>
	<li>
Raccolta di succo del midgut
	<ol>
<li>Anestetizzare gli scarafaggi sul ghiaccio. Sistemare gli scarafaggi lato ventrale sulla piastra di dissezione con freddo 1x insetto Salina buffer mediante spilli entomologici.</li>
		<li>Sezionare l'addome con una pinzetta bene. Rimuovere l'intestino intero (compreso l'intestino anteriore, media e posteriore) per un piatto fresco con 1 x insetto tampone salino.</li>
		<li>Rimuovere l'intestino anteriore e posteriore e di trasferire velocemente midgut ad un tubo del microcentrifuge che contiene 100 µ l di tampone salino dell'insetto.</li>
		<li>Piscina l'intestino da sei scarafaggi nel tubo stesso e vortexare per 10 s.</li>
		<li>Centrifugare per 10 minuti a 1.000 x g a 4 ° C per rallentare gli emociti e tessuti di intestino. Trasferire il surnatante in una provetta pulita microcentrifuga.</li>
		<li>Quantificare la concentrazione di proteine totali utilizzando uno spettrofotometro UV-Vis di micro-volume misurando A28015.</li>
		<li>Regolare ogni campione per avere la stessa concentrazione di proteina (6 mg / µ l di proteina totale).</li>
	</ol>
</li>
</ol>4. dsRNA degradazione Assay
<ol><li>Appena preparare 4 µ l del dsRNA nudo o lipoplessi dsRNA (1 µ g).</li>
	<li>Mescolare il succo di dsRNA enzimi di soluzioni con 10 µ l di tampone salino dell'insetto (controllo), estratti dall'emolinfa, o del midgut.</li>
	<li>Aggiungere 2 µ l di EGTA (20 mM) o acqua RNAsi-libera per separare campioni come controlli per inibizione delle attività enzimatiche.</li>
	<li>Incubare per 1 ora o più a lungo (6, 12 o 24 ore) a 25 ° C.</li>
	<li>Aggiungere 200 µ l di reagente di estrazione e cloroformio 40 µ l, poi vortice.</li>
	<li>Centrifugare per 10 min a 13.000 x g a 4 ° C, quindi raccogliere il surnatante (150 µ l) in un nuovo tubo del microcentrifuge.</li>
	<li>Precipitare dsRNA aggiungendo 150 µ l isopropanolo e incubare per 15 minuti in ghiaccio.</li>
	<li>Centrifugare per 15 min a 13.000 x g a 4 ° C, quindi rimuovere il surnatante.</li>
	<li>Aggiungere 200 µ l di etanolo 70% per lavare dsRNA pellet. Centrifugare per 10 min a 13.000 x g a 4 ° C.</li>
	<li>Rimuovere l'etanolo e ripetere le fasi di lavaggio di etanolo.</li>
	<li>Rimuovere l'etanolo e asciutto dsRNA pellet da concentratori centrifughi vuoto per 3 min. Risospendere dsRNA con acqua RNAsi-libera di 10 µ l.</li>
	<li>Verificare l'integrità del dsRNA Trattato tramite elettroforesi su gel di agarosio 1.5%.</li>
</ol>5. la somministrazione orale di dsRNA
<ol><li>Mantenere gli scarafaggi su una dieta ad libitum di cibo secco (chow cane). Privare l'approvvigionamento idrico dagli scarafaggi un giorno prima gli esperimenti di ingestione di dsRNA.</li>
	<li>Tenere uno scarafaggio afferrando le ali con le pinze flessibili (Figura 1, punto 5). Nota: Non afferrare le parti del corpo di scarafaggi.</li>
	<li>Preparare il dsRNA lipoplessi, come pure di dsRNA nuda, per l'alimentazione, come descritto nella sezione 2. Nota: Il dsRNA lipoplessi devono essere preparata fresca prima della poppata.</li>
	<li>Alimentazione 4 µ l di dsRNA lipoplessi o nudo dsRNA (250 ng) con una micropipetta.</li>
	<li>Lentamente dispensare la goccia della soluzione dsRNA vicino all'apparato boccale di scarafaggi e lasciare che gli scarafaggi ingerire la goccia completamente.</li>
	<li>Eseguire gli esperimenti di ingestione due volte al giorno (1 h dopo luci su e 1 h prima luci spente) per 8 giorni o 16 giorni di continuo. Nota: Tutte le blatte acquisito acqua solo tramite alimentazione manuale con reagenti soluzione o controllo di dsRNA (ad es., acqua gratuita nucleasi, soluzione di glucosio e reagente liposoma) durante gli esperimenti di ingestione.</li>
	<li>Fornire bottiglie d'acqua per gli scarafaggi dopo 16 giorni di ingestione di dsRNA esperimenti.</li>
</ol>6. valutare atterramento
<ol><li>Al momento scelto punti (2, 9 e 17 giorni dopo il test di ingestione di dsRNA), raccogliere gli insetti dsEGFP - e dsTub-trattati e sezionare i tessuti selezionati (per esempio, del midgut).</li>
	<li>Eseguire PCR quantitativa in tempo reale (qRT-PCR). Le condizioni di amplificazione qRT-PCR e informazioni sul primer come riportato da Lin et al. (2017) 12.</li>
	<li>Valutare il controllo e il dsRNA-Trattato insetti ogni giorno per controllare la mortalità e rimuovere gli scarafaggi che sono più in movimento.</li>
</ol>

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Questo protocollo presenta un metodo per RNAi efficace attraverso la consegna orale di dsRNA lipoplessi, che comprende la protezione contro la digestione di ribonucleasi nel succo del midgut il tedesco scarafaggio. Come dimostrato in altri studi in varie specie di insetti, l'effetto di RNAi povero attraverso la consegna orale di dsRNA è rappresentato principalmente dalla degradazione di dsRNA8,9,10. Questo protocollo produce li...

RNAi è stato dimostrato come un metodo efficace per l'espressione genica atterramento attraverso un meccanismo di silenziamento post-trascrizionale via innescato da molecole di dsRNA in molti eucarioti1. Nell'ultimo decennio di studi, RNAi è diventata uno strumento utile per studiare le funzioni dei geni dallo sviluppo di comportamento che riducono l'espressione di specifici geni tramite iniezione e/o alimentazione di dsRNA in vari taxa di insetti2,3. A causa della specificità e robustezza dell'effetto di svuotamento, l'applicazione di RNAi è attualmente considerato come una strategia potenziale per pest control gestione4,5. Tuttavia, l'efficienza di RNAi varia ampiamente tra specie di insetti, a seconda di diversi geni presi di mira e i metodi di consegna. Un corpo crescente di prova suggerisce che l'instabilità del dsRNA, che è stato degradato dalla ribonucleasi, è un fattore critico per la limitata efficacia della RNAi5,6. Per esempio, la bassa sensibilità di RNAi in Manduca sexta è stata spiegata dal fatto che il dsRNA mescolato con l'emolinfa è stato rapidamente degradato entro 1 ora7. Allo stesso modo, la presenza di nucleasi alcaline in midgut, che degradano in modo efficiente dsRNA ingerito, è fortemente correlata con bassa efficienza di RNAi in diversi ordini di insetti8,9,10.
La somministrazione orale di dsRNA è particolarmente interessante per l'applicazione del RNAi in una strategia di controllo dei parassiti, ma un metodo per ritardare la degradazione di dsRNA di nucleasi a midgut non è ancora stato sviluppato, che avrebbe il potenziale per garantire un efficace RNAi attraverso l'alimentazione. Tuttavia, il problema del blocco di RNAi per consegna orale di dsRNA è stato segnalato dalla grande quantità di dsRNA, ad es. 50 µ g in Bombyx mori, o alimentazione alimentazione continuamente per 8 giorni (8 µ g dsRNA in totale) nelle specie di locusta. Il tedesco scarafaggio, germinica di Blattella, è altamente sensibile alla RNAi tramite l'iniezione del dsRNA11,12,13,14, ma non è rispondente a dsRNA attraverso l'alimentazione. Recentemente, Lin et al. (2017) hanno dimostrato che il dsRNA incapsulato con risultati di vettori liposomi in successo RNAi per atterramento l'espressione genica α-tubulina nella mortalità significativa del midgut e trigger del tedesco scarafaggio15. Come la degradazione di dsRNA in midgut è il fattore limitante per RNAi orale, i vettori di liposomi servono come veicolo di proteggere dsRNA dalla degradazione, che è facilmente applicabile in altri insetti con le attività della nucleasi forte nell'intestino. Della nota, la ragione per la scelta del reagente di transfezione particolare (Vedi Tabella materiali) abbiamo usato come vettore di liposomi nel protocollo attuale è che è stato testato per la transfezione di cellule di insetto linea con meno tossicità, secondo la istruzioni del produttore. Secondo il confronto dei sistemi di transfezione diversi liposoma Gharavi et al. (2013) 16, l'efficienza di trasfezione Short interfering RNA (siRNA) è circa la stessa tra questo e altri sistemi disponibili in commercio che sono stati utilizzati per sistemi di dsRNA in altri insetti17,18 . Inoltre, il nostro metodo di alimentazione sia abbastanza attento a garantire la corretta quantità di dsRNA è ingerita da ogni scarafaggio, e che i risultati siano robusti e confermato. In sintesi, il presente protocollo e i risultati dimostrano che utilizzando dsRNA lipoplessi migliora la stabilità di dsRNA e apre la porta alla progettazione della consegna orale strategia di RNAi, che è un approccio promettente per controllo dei parassiti in futuro.

<table><tbody><tr><td>GenJe Plus DNA in vitro Transfection reagent</td><td>SignaGen</td><td>SL100499</td><td>for lipoplexes preparation</td></tr><tr><td>Blend Taq plus</td><td>TOYOBO&amp;nbsp;</td><td>BTQ-201</td><td>for PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ABI&amp;nbsp;</td><td>4385612</td><td>for qPCR</td></tr><tr><td>FirstChoice RLM-RACE Kit</td><td>Invitrogen</td><td>AM1700</td><td>for 3&#039; UTR identification&amp;nbsp;</td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Invitrogen</td><td>AMB13345</td><td>for dsRNA synthesis</td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2239</td><td>remove DNA template from dsRNA</td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596018</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>RQ1 RNase-Free Dnase</td><td>Promega</td><td>M6101</td><td>remove DNA template from total RNA&amp;nbsp;</td></tr><tr><td>chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2432</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>I9516</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>24102</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Diethyl pyrocarbonate, DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>for RNase free water preparation</td></tr><tr><td>glucose solution</td><td>Sigma-Aldrich</td><td>G3285</td><td>for lipoplexes preparation</td></tr><tr><td>Sodium chloride, NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>insect saline buffer formula</td></tr><tr><td>Potassium chloride, KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>insect saline buffer formula</td></tr><tr><td>Calcium chloride, CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016</td><td>insect saline buffer formula</td></tr><tr><td>Magnesium chloride hexahydrate, MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670</td><td>insect saline buffer formula</td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E3889</td><td>enzyme inhibitor&amp;nbsp;</td></tr><tr><td>dissecting scissor</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>fine tweezers</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>flexible tweezer</td><td>F.S.T.</td><td></td><td>cockroach holding&amp;nbsp;</td></tr><tr><td>pipetman</td><td>RAININ</td><td>P10</td><td>sample preparation</td></tr><tr><td>microcentrifuge tube</td><td>Axygen</td><td>MCT175C, PCR02C</td><td>sample preparation</td></tr><tr><td>pipette tip&amp;nbsp;</td><td>Axygen</td><td></td><td>sample preparation</td></tr><tr><td>vortexter</td><td>Digisystem</td><td>vm1000</td><td>sample preparation</td></tr><tr><td>Minispin centrifuge</td><td>The Gruffin Group</td><td>GMC 206</td><td>for liquid spin down&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>ALC</td><td>PK121R</td><td>sample preparation</td></tr><tr><td>pH meter&amp;nbsp;</td><td>JENCO</td><td>6071</td><td>for pH adjust</td></tr><tr><td>micro-volume spectrophotometer</td><td>Quawell</td><td>Q3000</td><td>nucleic acid quantitative</td></tr><tr><td>PCR Thermal cycler</td><td>ABI&amp;nbsp;</td><td>2720</td><td>for template PCR or&amp;nbsp; dsRNA synthesis incubation&amp;nbsp;</td></tr><tr><td>quantitative real-time PCR</td><td>ABI&amp;nbsp;</td><td>StepOne plus</td><td>gene expression quantitative</td></tr><tr><td>Centrifugal Vacuum Concentrators</td><td>eppendorf</td><td>5301</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Multipette&amp;nbsp;</td><td>eppendorf</td><td>xstream</td><td>for real-time PCR sample loading&amp;nbsp;</td></tr><tr><td>Agarose I</td><td>amresco</td><td>0710</td><td>for nucleic acid electrophoresis</td></tr><tr><td>tub gene specfifc forward preimer</td><td>tri-I biotech</td><td></td><td>GGG ACA AGC CGG AGT GCA GA</td></tr><tr><td>tub gene specfifc reverse preimer</td><td>tri-I biotech</td><td></td><td>TCC TGC TCC TGT CTC GCT GA</td></tr><tr><td>dsTub template forward primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&amp;nbsp;</td></tr><tr><td>dsTub template reverse primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&amp;nbsp;</td></tr><tr><td>dsEGFP template forward preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td></tr><tr><td>dsEGFP template reverse preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td></tr><tr><td>tub qPCR forward primer</td><td>tri-I biotech</td><td></td><td>GGA CCG CAT CAG GAA ACT GGC</td></tr><tr><td>tub qPCR reverse preimer</td><td>tri-I biotech</td><td></td><td>CCA CAG ACA GCC TCT CCA TGA GC</td></tr><tr><td>ef1 qPCR forward primer</td><td>tri-I biotech</td><td></td><td>CGC TTG AGG AAA TCA AGA AGG A</td></tr><tr><td>ef1 qPCRreverse preimer</td><td>tri-I biotech</td><td></td><td>CCT GCA GAG GAA GAC GAA G</td></tr></tbody></table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

Interferenza del RNA (RNAi) è stato ampiamente applicato per scoprire le funzioni biologiche di numerosi geni ed è stata prevista come un parassita controllo strumento operativo tramite rottura di espressione genica essenziale. Sebbene diversi metodi, come l'iniezione, alimentazione e ammollo, sono stati segnalati per il recapito corretto del RNA double-stranded (dsRNA), l'efficienza di RNAi attraverso la consegna orale di dsRNA è altamente variabile tra i diversi gruppi degli insetti. Il tedesco scarafaggio, Blattella germanica, è altamente sensibile all'iniezione del dsRNA, come dimostrato da numerosi studi pubblicati in precedenza. Il presente studio descrive un metodo per dimostrare che il dsRNA incapsulato con vettori liposoma è sufficiente per ritardare la degradazione di dsRNA dal succo del midgut. In particolare, l'alimentazione in continuo di dsRNA incapsulato da liposomi significativamente riduce l'espressione di tubulina in midgut e ha portato alla morte di scarafaggi. In conclusione, la formulazione e l'utilizzo di dsRNA lipoplessi, che proteggono il dsRNA contro nucleasi, potrebbe essere in futuro un uso pratico di RNAi per controllo dei parassiti dell'insetto.

Uno schema semplificato del protocollo per la somministrazione orale di dsRNA è presentato nella Figura 1, dove sono mostrati i passaggi chiave per la preparazione di dsRNA lipoplessi.
Al fine di indagare la protezione fornita dai vettori liposomi al degrado di dsRNA nel succo del midgut di b. germanica, un'analisi ex vivo in cui si svolse la dsTub lipoplessi sono state incubate c...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Uso pratico di interferenza del RNA: consegna orale del RNA Double-stranded in elementi portanti di liposomi per scarafaggi

Questo manoscritto viene illustrato lo svuotamento dell'espressione genica in midgut il tedesco scarafaggio attraverso ingestione orale di RNA double-stranded incapsulato in liposomi.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

Research

JoVE Journal

Biology

9.3K Views.  National Taiwan University. Questo manoscritto viene illustrato lo svuotamento dell'espressione genica in midgut il tedesco scarafaggio attraverso ingestione orale di RNA double-stranded incapsulato in liposomi.

Video: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Ricerca

Genetica

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Metodi di consegna orale RNA double-stranded per indurre RNAi nel floema e insetti Pentatomomorfi pianta-sap-alimentazione

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Ambiente

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Riproducibile dsRNA Microinjection e analisi biologica di deposizione delle uova di zanzare e mosche domestiche

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

Interferenza dell'RNA nei Coleotteri acquatici come potente strumento per manipolare l'espressione genica in punti specifici del tempo di sviluppo

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Applicazioni dell'interferenza dell'RNA nello scarafaggio americano

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

Biologia dello sviluppo

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 ...

Biologia

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Il chitosano / interferenti RNA nanoparticelle mediata silenziamento genico in Disease Vector Mosquito Larve

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

Somministrazione efficace di interferenza orale dell'RNA (RNAi) alle zanzare Anopheles gambiae adulte

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

<meta charset="utf8"></head><body>Alimentazione del dsRNA mediata da nanoparticelle per il silenziamento genico nelle larve del baco da seta

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.

<meta charset="utf8"></head><body>Autore in primo piano: Avanzamento della ricerca sul silenziamento genico nei bachi da seta con somministrazione di dsRNA attraverso l'alimentazione di nanopartice

Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.

Interferenza dell'RNA larvale nel baco da seta Bombyx mori attraverso la somministrazione di nanoparticelle di chitosano/dsRNA

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

Uso pratico di interferenza del RNA: consegna orale del RNA Double-stranded in elementi portanti di liposomi per scarafaggi

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Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,