Name: genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy
Uploaded: 2018-04-03T13:00:00
Description: Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

Dette arbejde blev støttet af tilskud fra Ontario Institut for kræftforskning, Canada Foundation for Innovation, Ottawa regionale Cancer Foundation og Terry Fox Research Institute. K.J.A. blev støttet af en Vanier Canada Graduate stipendium, canadiske Institut for sundhed Research-Master's Award, og en Ontario Graduate stipendium.

1. udvikling og validering af en høj overførselshastighed siRNA screening assay
Bemærk: For alle eksperimenter, brug Hepes-buffered vækst medier hensigtsmæssigt for hver cellelinje. Se figur 1 for en oversigt over arbejdsproces fra assay optimering til tertiær validering.
<ol><li>Bestemmelse af de optimale Transfektion betingelser
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<li>Vælg en tumor cellelinje eller ideelt set flere tumor celler linjer til at optimere Transfektion be...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Median+absolute+deviation+to+improve+hit+selection+for+genome-scale+RNAi+screens.">Median absolute deviation to improve hit selection for genome-scale RNAi screens.</a> J. Biomol. Screen. 13 (2), 149-158 (2008).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+Score,+SSMD*,+z*+Score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z Score, SSMD*, z* Score, and t statistic for hit selection in RNAi high-throughput screens.</a> J. Biomol. Screen. 16 (7), 775-785 (2011).</li><li>Zhang, X. D., Lacson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+SSMD-based+false+discovery+and+false+nondiscovery+rates+in+genome-scale+RNAi+screens.">The use of SSMD-based false discovery and false nondiscovery rates in genome-scale RNAi screens.</a> J. Biomol. Screen. 15 (9), 1123-1131 (2010).</li><li>Amberkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNA+interference+screens+integrative+analysis:+towards+a+comprehensive+understanding+of+the+virus-host+interplay.">High-throughput RNA interference screens integrative analysis: towards a comprehensive understanding of the virus-host interplay.</a> World J. Virol. 2 (2), 18-31 (2013).</li><li>Barrows, N. J., Le Sommer, C., Garcia-Blanco, M. A., Pearson, J. 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Her præsenterer vi en protokol for at ansætte høj overførselshastighed RNAi screening at identificere vært mål, der kan manipuleres til at forbedre oncolytic virus terapi. Dette er blevet testet med succes med oncolytic Maraba virus og oncolytic vaccinia virus, men, som bemærket, det kan tilpasses til brug med andre oncolytic vira eller andre vira generelt for at identificere vært gener, at modulerer virus replikering. Screening protokol er også designet til at identificere gener, at øge eller mindske spredning...

Høj overførselshastighed genome-wide RNAi screeningen har vist sig for at være et uvurderligt værktøj for afslørende biologiske indsigt i forskellige områder af undersøgelse, herunder virus biologi. Over det seneste årti eller deromkring, har talrige grupper gennemført cellebaserede storstilet RNAi screening for at udstrakt grad identificere vært gener, at modulerer virus replikation (anmeldt i1,2,3,4 , 5 , 6 , 7). interessant, et stort antal af disse skærme er blevet ført i kræftceller og flere med vira, der er aktuelle oncolytic virus kandidater herunder vaccinia virus8,9,10 , Myxom virus11, herpes simplex virus12, vesikulær stomatitis-virus13,14, og Maraba virus15. Mens størstedelen af disse undersøgelser fokuserede på at identificere vært faktorer, der påvirker nogle aspekter af virus replikering og ikke identificerer biotherapeutic mål for at øge oncolytic virus terapi, kunne værdifulde data udvindes fra disse datasæt i denne henvisning. Det er klart, at den kraftfulde potentiale til at identificere vært mål, der kan manipuleres til at forbedre oncolytic virus terapi ikke er blevet fuldt udnyttet.
En overflod af oncolytic virus platforme testes i øjeblikket i prækliniske og kliniske undersøgelser herunder herpes simplex virus, reovirus og vaccinia virus, som har haft påviselige succes i sene kliniske forsøg. For fleste af disse platforme, har indsats været rettet mod at ændre virus genomer at forbedre terapi. For eksempel, for at øge tumor specificitet, er specifikke virus gener blevet slettet eller muteret betydeligt begrænser virusreplikation i normale celler, men ikke i tumorceller. I nogle tilfælde transgener er blevet tilføjet som GM-CSF (granulocyt makrofag koloni-stimulerende faktor) til at forstærke immunresponset eller NIS (natrium Iodid symporter) til at aktivere in vivo billeddannelse og cellulære radioiodide optagelse terapeutisk formål. Der har dog været meget lidt arbejde fokuseret på at manipulere vært/tumor gener og udforske virkningen af host/tumor gener på oncolytic virus replikering. Med fremkomsten af high throughput screening teknologi, er det nu muligt at sonde vært-virus interaktioner i genom skala og afkode muligheder for at manipulere vært genom for at forbedre oncolytic virus terapi (gennemgik i16, 17,18).
Vi rapporterede den første undersøgelse demonstrerer proof-of-concept for at bruge høj overførselshastighed genetisk screening for at identificere vært mål, at kunne manipuleres til at forbedre oncolytic virus terapi. For at opdage vært gener, at modulerer Maraba virus oncolysis, en oncolytic rhabdovirus i øjeblikket bliver testet i fase I og II forsøg, vi gennemførte genome-wide RNAi skærme på tværs af tre forskellige tumor cellelinjer i to eksemplarer med en siRNA (kort blander RNA) biblioteket målretning 18,120 gener15. Sti analyse af hits identificeret i skærmene viste berigelse af medlemmer af den ER (endoplasmatiske reticulum) stress reaktion veje. Vi har valgt 10 hits inden for disse veje for sekundære validering ved hjælp af siRNA med forskellige målretning sekvenser fra dem af den primære skærm. Som en del af tertiære validering, gennemførte vi en redning eksperiment med IRE1α (inositol-kræver enzym 1 alpha) til at bekræfte, at ramt var på målet. In vitro og i vivo test ved hjælp af en lille molekyle hæmmer af IRE1α resulterede i drastisk forbedret oncolytic effektivitet.
Workenhe et al. 12 har også gennemført en genome-wide RNAi skærmen ved hjælp af en samlet lentiviral shRNA (kort hårnål RNA) bibliotek målretning 16,056 gener for at identificere vært faktorer begrænser den menneskelige herpes simplex virus type 1 (HSV-1) mutant KM100-medieret oncolysis af brystkræft kræftceller. I den primære skærm identificeret de 343 gener knockdown af som fører til øget cytotoksicitet af KM100-inficerede celler over mock-inficerede celler; de udvalgt 24 af disse gener for sekundære validering. Ud af de 24 gener, 8 gener blev bekræftet i den sekundære skærm med en af dem er SRSF2 (Serin/arginin-rige splejsning faktor 2), som efterfølgende blev bekræftet ved hjælp af en genetisk rescue eksperiment under tertiære validering. Gruppen fortsatte med at identificere en DNA topoisomerase hæmmer kemoterapeutiske at reduceret fosforylering af SRSF2 og viste, at kombinationsbehandling af HSV-1 KM100 med dette hæmmer fører til forlænget overlevelse af TUBO tumor-bærende mus.
I de ovennævnte undersøgelser fulgte en lignende arbejdsproces. Hver begyndte med en primær genome-wide RNAi skærmen efterfulgt af en sekundær skærm på et udvalgt antal af hits identificeret i den primære skærm. Dette blev efterfulgt af tertiære valideringsundersøgelser, som omfattede bekræfter, at ramt var på mål ved at udføre genetiske redde forsøg in vitro med ultimative validering udføres ved at manipulere target gen produktet in vivo. I denne artikel give vi først en generel protokol til udvikling og validering af en høj overførselshastighed siRNA-screening assay, som indebærer fastsættelse af optimale Transfektion betingelser, mængden af virus, og længden af infektion. Vi tilbyder også en detaljeret protokol til at foretage en primær en høj overførselshastighed siRNA skærm samt en samlet beskrivelse af metoden til udførelse af sekundær skærm validering og tertiære validering.

<table>
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA library</td>
			<td>GE Dharmacon</td>
			<td>G-005005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 384-well plate</td>
			<td>Becton Dickinson</td>
			<td>353962</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome SMARTpool PLK1</td>
			<td>GE Dharmacon</td>
			<td>M-003290-01</td>
			<td></td>
		</tr>
		<tr>
			<td>AllStars Negative Control siRNA</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome Non-targeting siRNA Pool #2</td>
			<td>GE Dharmacon</td>
			<td>D-001206-14-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/HIGH Glucose</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F15051-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution (1M)</td>
			<td>Sigma</td>
			<td>H3535-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X solution</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2500056</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligofectamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>1225011</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma</td>
			<td>R7017-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (37% by weight)</td>
			<td>Fisher Scientific</td>
			<td>F79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Corning</td>
			<td>430282</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Sealing Film For Microplates</td>
			<td>Excel Scientific</td>
			<td>361006007</td>
			<td></td>
		</tr>
		<tr>
			<td>Peelable Heat Sealing Foil</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3720</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek MicroFlo Select Dispenser cassette</td>
			<td>Fisher Scientific</td>
			<td>11-120-625</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFlo Select (liquid dispenser)</td>
			<td>Fisher Scientific</td>
			<td>11120621</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HT plate reader</td>
			<td>Biotek</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera Imaging and Analysis Instrument</td>
			<td>PerkinElmer</td>
			<td>HH10000115</td>
			<td></td>
		</tr>
		<tr>
			<td>KiNEDx Robotic Arm</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>KX-300-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytomat 24C Series Automated Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>50080227</td>
			<td></td>
		</tr>
		<tr>
			<td>JANUS Automated Workstation</td>
			<td>PerkinElmer</td>
			<td>AJI4M01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Carousel</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Alps 3000 plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy

Høj overførselshastighed genome-wide RNAi (RNA interferens) screening teknologi har været meget anvendt til at opdage vært faktorer, der påvirker virus replikering. Her præsenterer vi anvendelsen af denne teknologi til at afdække vært mål, som specifikt modulerer replikering af Maraba virus, en oncolytic rhabdovirus og vaccinia virus med mål at forbedre terapi. Mens protokollen er blevet testet til brug med oncolytic Maraba virus og oncolytic vaccinia virus, denne fremgangsmåde er gældende for andre oncolytic virus og kan også udnyttes til at identificere vært mål, at modulerer virus replikering i pattedyrsceller i generelle. Denne protokol beskriver udviklingen og valideringen af en analyse for høj overførselshastighed RNAi screening i pattedyrceller, centrale overvejelser og forberedelsestrin vigtigt for at gennemføre en primær høj overførselshastighed RNAi skærm og en trinvis vejledning til gennemføre en primær høj overførselshastighed RNAi skærm; Derudover beskriver det bredt metoder til at foretage sekundær skærm validering og tertiære valideringsundersøgelser. Fordelen ved høj overførselshastighed RNAi screening er, at det tillader en at katalogisere, i en omfattende og upartisk måde, vært faktorer, som modulerer ethvert aspekt af virus replikering, kan man udvikle en in vitro- assay som smitteevne, brast størrelse, og cytotoksicitet. Det har beføjelse til at afdække biotherapeutic mål uforudsete baseret på aktuelle viden.

En oversigt over arbejdsgangen for at identificere vært mål for at forbedre oncolytic virus terapi er præsenteret i figur 1.
Som illustreret i figur 1, er den kritiske første skridt til at gennemføre en høj overførselshastighed RNAi skærm assay udvikling. Figur 2A giver en prøve plade layout for Transfektion optimering assay. <stro...

Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

Genome-wide RNAi Screening at identificere vært faktorer, at modulerer Oncolytic Virus terapi

Her beskriver vi en protokol for at ansætte høj overførselshastighed RNAi screening at afdække vært mål, der kan manipuleres til at forbedre oncolytic virus terapi, specielt rhabodvirus og vaccinia virus behandling, men det kan være let tilpasses til andre oncolytic virus platforme eller for at opdage vært gener, at modulere virus replikering generelt.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

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