Name: rescue of recombinant newcastle disease virus from cdna
Uploaded: 2013-10-11T23:30:00
Description: Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Forfatterne vil gerne takke tidligere og nuværende medlemmer i laboratorier Drs. Peter Palese og Adolfo García-Sastre for udviklingen af ​​NDV omvendt genetik teknikker og for teknisk assistance. Forskning i Newcastle disease-virus i AG-S laboratorium er delvist finansieret af niaD indrømme R01AI088770 og af Department of Homeland Security Science &amp; Technology Center of Excellence for Emerging og zoonotiske Dyresygdomme (CEEZAD, tildeling nummer 2010-ST-061-AG001). Forskning i LM-S laboratorium er finansieret af NIH tilskud RO1 AI077719, R21NS075611-01, R03AI099681-01A1, NIAID Centers of Excellence for Influenza Forskning og overvågning (HHSN266200700008C) og University of Rochester Center for Biodefense Immun Modeling (HHSN272201000055C) .

1.. Fremstilling af pattedvrceller (figur 4A, dag 1) Split HEp-2-eller A549-celler dagen før transfektion i 6-brønds plader. Tæthed af cellerne skal nå 80-90% konfluens den følgende dag. Normalt kan en konfluent 100 mm skål opdeles i 8 brønde (ca. 1 x 10 6 celler pr brønd). For hver virus der skal reddes, bør 2-4 forskellige brønde medtages, samt 2 ekstra brønde til kontrollerne pCAGGS-GFP-og pCITE-GFP-18, der har til formål at overvåge trans...

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Flere faktorer skal tages i betragtning for at opnå gode resultater, mens redde NDV. For det første fuldlængde-cDNA-konstruktion, der skal anvendes skal være udformet således, at den funktionelle integrering af de nye transgener / ændringer i NDV-genomet. Det betyder, som nævnt ovenfor, at (i) passende gen ende (GE), intergen (IG) og gen-start (GS) sekvenser der skal tilføjes, hvis det kræves, (ii) der er ingen formodede GE eller GS sekvenser i udenlandske gen, og (iii) den fulde rekombinante genom følger de &...

Newcastle disease-virus (NDV), en aviær paramyxovirus tilhører Avulavirus slægten 1, er en økonomisk relevant og dermed i vid udstrækning forsket og overvåget zoonotisk agens, der kan alvorligt påvirke fjerkræavl hele verden. Selvom det ikke er et humant patogen, NDV er også blevet grundigt undersøgt ud over dyrlægen felt både som model paramyxovirus og på grund af dens meget interessante, naturlige onkolytiske egenskaber 6. Forskning i NDV høj grad nydt godt af udviklingen af teknikker til omvendt genetik til enkelt-strandede, ikke-segmenterede negativ-sense RNA-virus, først beskrevet for rabies virus ved Conzelmann og coleagues 2. En række af genetisk modificerede NDVs, bærer fremmede gener eller modifikationer af deres vildtype genom er blevet bredt studeret lige siden. Arbejdet med disse rekombinante vira har været afgørende for at karakterisere forskellige virulensfaktorer ikke kun for NDV, men også af andre relevante humanin patogener såsom influenza A virus 7 - eller den emergente Nipah virus 8. Endvidere har en række forskellige undersøgelser undersøgt brugen af disse teknikker til at forbedre den medfødte antitumoraktivitet af NDV 6,9,10, for det meste ved at styrke de immunstimulerende egenskaber af viruset. Anden relevant forskningsområde på rekombinante NDVs har været generation af vaccinekandidater mod andre virussygdomme som influenza 5,11,12, HIV 13, mæslinger 14, SARS 15, eller at forårsaget af respiratorisk sincytial virus (RSV) 16. Blandt de forskellige bemærkelsesværdige fordele, som NDV er manglen på allerede eksisterende immunitet i befolkningsgrupper, stabiliteten af de udenlandske genetiske skær, manglende recombinatory aktivitet og samlet en høj sikkerhedsprofil kombineret med de førnævnte naturlige immunstimulerende egenskaber 17. Det er også bemærkelsesværdigt den potentielle anvendelse af rekombinante bivalente vacciner poultry, beskyttende mod både NDV og højpatogen aviær influenzavira 11,12. Dette kan være en glimrende måde at mindske chancerne for sidstnævnte breder sig fra vilde fugle til tamme dyr, og dermed også med til at forebygge en eventuel inter-specifik hoppe af den frygtede fugleinfluenza til mennesker. Endelig er reporter-udtrykkende NDV blevet anvendt til evaluering af medfødte immunresponser samt identifikation af interferon-antagonist, der kodes af flere vira 18-27. Den redningsproces af en rekombinant, ikke-segmenteret negativ-strenget RNA-virus består dybest set på kunstigt at tvinge en virale replikationscyklus i en producerende celle ved transfektion af cDNA, der koder for den minimale infektiøse molekylære maskineri, der er kendt som ribonukleoprotein eller RNP (figur 2). De RNP&#39;er består af den virale polymerase (P-og L-proteiner), nukleoprotein (NP) og fuldlængde antigenomiske RNA virus. Denne RNA + antigenom er the skabelon kræves til frembringelse af de komplementære RNA-genomer, som også er forbundet med resten af proteiner af viral RNP rekapitulerer samme smitsomme kompleks, et naturligt virus ville frigøre på cellens cytoplasma efter infektion (figur 2A ). Fra dette trin frem, kan den virale cyklus foregå naturligt og rekombinante virioner indkapsling de modificerede genomer, vil blive genereret (Fig. 2B). Bemærkelsesværdigt transfektion af cDNA i stedet for det antigenomiske cDNA stærkt indskrænker eller ophæver fuldstændigt redning effektivitet 2,28-30. Selv når antigenomiske cDNA transficeres, er effektiviteten af ​​indkapsling af det rekombinante RNA i RNP&#39;er i transficerede celler sandsynligvis meget lav. På grund af dette, rednings-protokoller for NDV omfatter ofte forskellige trin til amplifikation af de få virale partikler, der frigives fra de oprindeligt blev transficerede celler ved coculturing dem med permissive celler og / eller vedinfektion af embryonerede æg. Forud for redning, kan cDNA&#39;en manipuleres ved standard kloningsteknikker procedurer med henblik på at generere de ønskede modifikationer. Mens specifikke mutationer i de forskellige genprodukter og regulatoriske sekvenser af virus kan ligefrem måde opnås, mange af de offentliggjorte arbejde, der involverer rekombinant NDV har krævet tilsætning af en ny transkriptionsenhed i NDV-genomet. Ligesom andre medlemmer af paramyxovirus familie, NDV-genomet koder for otte forskellige proteiner i seks transkriptionsenheder, som udtrykkes differentielt afhængigt af deres placering i forhold til 3&#39;-enden i en faldende gradient kritisk for den virale livscyklus 1. På grund af dette, skal placeringen af ​​den nye transkriptionsenhed i genomet være nøje udvalgt for at opnå en balance mellem ekspressionen af ​​transgenet og nedskrivning af viral replikation. Indsættelse mellem P-og M-gener er blevet brugt mest, tHough andre steder er også blevet testet 13,31. Uanset indsatsen, kloning i NDV-cDNA skal følge nogle regler for at generere en rescuable konstruktion: (i) et nyt gen, der skal indbefattes i NDV-genomet skal være under kontrol af de passende signaler til den virale RNA-afhængige RNA- polymerase. Disse sekvenser skal tilføjes opstrøms for den nye åbne læseramme (ORF), så polymerasen kan genkende slutningen af ​​den foregående gen (GE) og begyndelsen af ​​det nye transgen (GS), adskilt af en enkelt intergenisk sekvens nukleotid (IG) . Tilsætning af en gyldig Kozak (K)-sekvens for at forbedre eukaryote ribosomale oversættelse anbefales også til bedre fremmed protein ekspression 32, (ii) en effektiv replikation af NDV, som for de fleste medlemmer af Paramyxoviridae-familien, er afhængig af genomet længde er multiplum af seks 33, og derfor enhver indsættelse i NDV skal følge denne &quot;regel af seks&quot;. Hvis det er nødvendigt, required yderligere nukleotider kan tilsættes nedstrøms den nye ORF, og (iii) sekvensen af ​​transgenet bør kontrolleres for at finde mulige GE og GS lignende sekvenser, som kan påvirke redning effektivitet, transgen ekspression og / eller virus levedygtighed. Hvis til stede, skal disse sekvenser fjernes ved stum mutagenese. Fremstillingen af ​​rekombinant fuld længde cDNA efter ovennævnte regler er det første skridt med henblik på effektivt at producere en genetisk modificeret NDV som beskrevet her. I systemet alle DNA-konstruktioner under kontrol af T7-RNA-polymerase-promotor (figur 3). Denne cytoplasmatisk polymerase tilvejebringes i trans ved coinfektion med et rekombinant modificeret vaccinia Ankara-virus (MVA-T7) 34 Figur 3A viser pNDV-B1 plasmid, der koder for fuldlængde-antigenomiske cDNA 5.. 3B viser pTM1 plasmider kodende for NP, P-og L ORF&#39;er. Plasmider pCITE-GFP, som koder for ufter T7 promotoren, grønt fluorescerende protein (GFP) og pCAGGS GFP 18, som koder for den samme ORF under chicken beta-actin-promotor 35, anvendes som kontroller. I denne protokol viser vi procedure at redde rekombinant NDV fra cDNA&#39;et af lentogene NDV-stamme Hitchner B1 5 (figur 4).

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

Newcastle disease-virus (NDV), prototypen medlem af Avulavirus slægten af familien Paramyxoviridae 1, er en ikke-segmenteret, negative-sense, single-strandede, kappeklædte RNA-virus (figur 1) med potentielle anvendelser som en vektor til vaccination, og behandling af humane sygdomme. Dybdegående udforskning af disse applikationer er kun blevet muligt efter oprettelsen af teknikker til omvendt genetik at redde rekombinante vira fra plasmider, der koder deres fuldstændige genomer som cDNA 2-5. Viral cDNA kan bekvemt modificeres in vitro ved anvendelse af standard kloningsprocedurer at ændre genotype af virus og / eller til at omfatte nye transkriptionsenheder. Redning af sådanne genetisk modificerede virus giver et værdifuldt redskab til at forstå faktorer, der påvirker flere stadier af infektionen, samt giver mulighed for udvikling og forbedring af vektorer til ekspression og levering af antigenertil vaccination og terapi. Her beskriver vi en protokol til redning af rekombinante NDVs.

Redning af NDV er en veletableret procedure rutinemæssigt udføres i laboratorier, der har adgang til den fuldstændige cDNA af virus. Den iboende stokastiske natur af fremgangsmåden gør det imidlertid vanskeligt at opnå 100% redning effektivitet. Overvågning de tidlige trin i processen, specielt transfektionseffektiviteten og infektionen med MVA-T7, hjælper at identificere mulige problemer. 5A viser standard transfektion og transfektion / infektion effektivitetsgevinster, der er nok til en vellyk...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease-virus (NDV) er blevet grundigt undersøgt i de seneste år for at udvikle nye vektorer til vaccination og behandling, blandt andre. Disse undersøgelser har været mulig på grund af teknikker til at redde rekombinant virus fra cDNA, som dem vi beskriver her.

Redning af rekombinant Newcastle Disease-virus fra cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

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