Name: production and titering of recombinant adeno-associated viral vectors
Uploaded: 2011-11-27T14:00:00
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Description: Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Se figur 1 for en illustrasjon oppsummere følgende protokollen. Sikkerhet Merk: Alt materiale som har vært i kontakt med montert viruspartikler må desinfiseres med Virkon-løsning eller annet egnet desinfeksjonsmiddel. 1. Utarbeidelse av plasmid DNA aksjer (~ 2 dager) <ol><li> Følgende plasmider kreves 1: <ul><li> pRV1 - som inneholder den AAV2 Rep og Cap sekvenser </li><li> pH21 - som inneholder den AAV1 Rep og Cap sekvenser </li><li> pFdelta6 - Adenovirus-helper plasmid </li><li> AAV plasmid som inneholder rekombinant uttrykket kassetten flankert av AAV2 emballasje signaler (invertert terminal gjentar, ITRs) </li></ul></li><li> Mens pFdelta6 bør dyrkes i Stbl2 kompetente celler for å unngå delvis sletting, kan pRV1 og pH21 dyrkes i DH5alpha kompetente celler. AAV plasmider kan dyrkes i Stbl2 celler hvis delvis slettinger skje i DH5alpha celler. </li><li>Plasmid DNA bør være av høy kvalitet og fri for RNA forurensninger. Plasmider kan screenes for integritet ved hjelp av følgende digests: <ul><li> pRV1 - fordøye med XbaI å gi band på 7,5 kb og 3,8 kb </li><li> pH21 - fordøye med EcoRI å gi band på 4,5 kb, 2,8 kb og 0,2 kb </li><li> pFdelta6 - fordøye med HindIII å gi band på 5,5 kb, 3 kb, 3 kb, 2,3 kb og 1,5 kb </li></ul></li></ol> 2. Utarbeidelse av humane embryonale Kidney 293 (Hek293) celler for transfeksjon (2 - 3 dager) <ol><li> Plate to 80% confluent 150 cm 2 flasker av Hek293 celler i fem 15 cm diameter Nunc vev kultur retter. Cellene skal være 70 - 80% confluent før transfeksjon (ca 48 timer etter plating). Kultur celler i standard Dulbecco modifiserte Eagle medium (DMEM) med lav glukose som inneholder 10% kalvefoster serum og 100 U / ml penicillin / 100 mikrogram / ml streptomycin. </li><li> 3 timer før transfeksjon fjerne DMEM og erstatte med Iscove&#39;S modifisert Dulbecco medium (IMDM) som inneholder 5% kalvefoster serum. </li></ol> 3. Transfeksjon av viral plasmider (~ 1 time for transfeksjon, 3 dager for inkubering) <ol><li> Forbered følgende for en enkelt batch (5 x 15 cm vev kultur plater) av virus: <ul><li> 62,5 mikrogram AAV plasmid </li><li> 125 mikrogram pFdelta6 </li><li> 31,25 mikrogram pRV1 </li><li> 31,25 mikrogram pH21 </li><li> 1650 mL 2,5 M CaCl 2 </li><li> 12 ml dH 2 O </li></ul></li><li> I en klasse 2 vevskultur hette sterile filtrere transfeksjon blandingen i en 50 ml tube. </li><li> Mens virvling løsningen, raskt legge til 13 ml av 2 x HEPES buffered saltvann (pH 7,05). Erstatt lokket på 50 ml tube og fortsette å vortex i 15 sekunder. La stå i 1 minutt 45 sekunder, bør et fint hvitt bunnfall form. </li><li> Tilsett 5 ml av transfeksjon løsning til hver 15 cm vev kultur parabolen. Swirl platene til mix og gå tilbake til inkubatoren. </li><li> 16 timer etter transfeksjon fjerne IMDM medium og erstatte med DMEM. </li></ol> Fire. Lysing av celler og høsting av rAAVs (2 timer) <ol><li> 72 timer etter transfeksjon, fjerne media fra cellekultur plater og kast. Alt avfall skal behandles med Virkon-løsning eller annet egnet desinfeksjonsmiddel. </li><li> Forsiktig vaske cellene i varmt 1x fosfatbufret saltløsning (PBS, pH 7,4). </li><li> Tilsett 25 ml varm PBS til hver tallerken og forsiktig fjerne celler med en celle skrape. Samle suspensjon i 50 ml rør. </li><li> Pellet cellene ved 800 xg i 10 minutter. </li><li> Kast supernatant og resuspender pellet i 150 mM NaCl, 20 mM Tris pH 8,0, bruk 10 ml per vev kultur plate. Delt inn i to 50 ml rør. </li><li> Forbered en frisk løsning av 10% natrium deoxycholate i dH 2 O. Legg til 1,25 ml av dette til hvert rør for en endelig konsentrasjon på 0,5%. Legg benzonase nukleasefritt til en endelig konsentrasjon på 50 enheter per ml. Bland tube grundig. </li><li&gt; Inkuber ved 37 ° C i 1 time. </li><li> Fjern mobilnettet avfall ved sentrifugering ved 3000 xg i 15 minutter. Transfer til frisk 50 ml tube, sikre at alle celleavfall har blitt fjernet for å hindre blokkering av heparin kolonner (se trinn 5). På dette stadiet, kan prøvene oppbevares ved -20 ° C før du fortsetter. Vi har lagret prøver i flere uker ved -20 ° C uten en reduksjon i smittsomhet. </li></ol> 5. Heparin kolonne rensing av rAAVs (2 - 3 timer) <ol><li> Oppsett HiTrap heparin kolonner ved hjelp av en peristaltiske pumpe slik at løsningene strømmer gjennom kolonnen ved 1 ml per minutt for skritt 05.02 til 05.04. Det er viktig å sikre at ingen luftbobler er innført i heparin kolonnen. </li><li> Likevekt kolonnen med 10 ml 150 mM NaCl, 20 mM Tris, 8,0 pH. </li><li> Bruk 50 ml virus løsning til kolonne og la det strømme gjennom. </li><li> Vask kolonnen med 20 ml 100 mM NaCl, 20 mM Tris, 8,0 pH. </li><li> Bruk en 5 ml sprøyte fortsette å vaske kolonnen wed 1 ml 200 mM NaCl, 20 mM Tris, 8,0 pH, etterfulgt av 1 ml 300 mM NaCl, 20 mM Tris, 8,0 pH. Forkaste gjennomstrømning. </li><li> Bruk 5 ml sprøyter og milde press Eluer viruset fra kolonnen ved å: <ul><li> 1,5 ml 400 mM NaCl, 20 mM Tris, 8,0 pH </li><li> 3,0 ml 450 mM NaCl, 20 mM Tris, 8,0 pH </li><li> 1,5 ml 500 mM NaCl, 20 mM Tris, 8,0 pH </li><li> Samle disse i en 15 ml sentrifugerør. </li></ul></li></ol> Seks. Konsentrasjon og sterile filtrering av rAAVs (1 time) <ol><li> Konsentrer vektor bruker Amicon ultra-4 sentrifugalfilter enheter med 100 000 molekylvekt cutoff. Load 4 ml av kolonne eluatet inn i konsentratoren og sentrifuger ved 2000 xg i 2 minutter (ved romtemperatur). Kast flowthrough og reload konsentratoren med gjenværende virus løsning og gjenta sentrifugering. Den konsentrerte volum bør være ca 250 mL. Hvis konsentrert volumet er betydelig mer enn dette, forkaste flow through og fortsette å sentrifuge i ett minutt trinn inntil volumet er ca 250 mL. </li><li> Tilsett 250 mL PBS til virus for en endelig volum på 500 mL og fjerne fra konsentratoren. </li><li> Filter vektor gjennom en 13 mm diameter på 0,2 mikrometer sprøyte filter. Vector bør alikvoteres og oppbevares ved -80 ° C inntil nødvendig. </li></ol> 7. Titering of viral aksjer (3 dager) Dette krever en promoter som er aktiv i Hek293 celler kjører en reporter gen eller en immunocytochemically detectable genet produktet. Ved produksjon av en vektor som ikke er kompatibel med Hek293 celler andre cellelinjer eller primære cellekulturer kan brukes. <ol><li> Forbered 18 poly-L-lysin belagt glass Dekkglass eller 18 brønner på Nunc kammer lysbilder ved seeding Hek293 celler slik at de er 40 - 50% confluent. For titrering av Cre-avhengige rAAVs bruke Hek293 celler som stabilt uttrykker Cre recombinase 2. </li><li> Infisere hver brønn med serienummer dilutions av AAV vektor (fig 2A). Bruk 3 brønner per fortynning. Vi rutinemessig legger viruset direkte til hver brønn. </li><li> Etter 72 timer fikse Hek293 celler ved å legge til et likt volum på 4% Paraformaldehyde (PFA) til medium (gi en endelig konsentrasjon på 2% PFA) i ti minutter ved romtemperatur. </li><li> Vask celle tre ganger i PBS, skyll i dH 2 O og dekkglass. </li><li> Tell antall transduced celler fra de tre brønnene som har høyest fortynningsfaktoren, men likevel inneholder infiserte celler, finne gjennomsnittet av disse og multipliserer med fortynningsfaktoren for å gi antall smittsomme enheter per mikroliter (Fig. 2B). </li></ol> 8. Forventede resultater Hek293 celler transduced med et virus koding forbedret grønt fluorescerende protein (eGFP) er vist i figur 2B. Vi generelt oppnå konsistente titere på rundt 6 x 10 6 infeksiøse partikler per mikro liter. Vi rutinemessig bruker disse vektorer for stereotaxic injection inn i den voksne gnager hjernen. Dette gir en kraftig teknikk for region-spesifikke genuttrykk 2. Å kombinere denne regionen spesifisitet med celle-type selektiv genuttrykk vi injisere Cre recombinase-avhengige rAAVs inn mål regioner av Cre-transgene mus 2. Figur 2C-E viser eksempler på stereotaxic injeksjoner av Cre-avhengige rAAVs koding eGFP inn i ulike områder av hjernen hos voksne parvalbumin-Cre transgene mus 3. <img alt="Figur 1" src="/files/ftp_upload/3348/3348fig1.jpg" /> Figur 1. Skjematisk illustrasjon av protokollen for rAAV produksjon. . 1) Hek293 celler er belagt og vokst til 70 - 80% Confluence. 2.) AAV og helper plasmider er forberedt og transfektert inn Hek293 celler. 3). Medium er endret tilbake til DMEM 16 timer etter transfeksjon og Hek293 celler dyrkes i ytterligere 56 timer. 4). Hek293 cellene er høstet og lyseres. rAAV vektorer er skilt fra celleavfall av centrifugation. 5). Viral partikler er renset gjennom heparin kolonner før konsentrasjon og sterilisering. 6). Hek293 cellene er dyrket på 24 godt plater og infisere med serielle fortynninger av AAV vektor for å bestemme antall smittsomme enheter. <img alt="Figur 2" src="/files/ftp_upload/3348/3348fig2.jpg" /> Figur 2. Titering og in vivo anvendelse av rAAV1 / 2. (A) Oppsett av Hek293 celler for måling av viral titer. Hek293 celler er transduced med serielle fortynninger av rAAVs. C, friske kontroll; N, 1 mL av ufortynnet viral lager. (B) Hek293 celler infisert med rAAVs uttrykke eGFP. (CE) Viral eGFP uttrykk er begrenset til Cre-uttrykke nevroner i ulike målgrupper regioner av parvalbumin-Cre transgene mus etter stereotaxic injeksjon av Cre-aktivert rAAVs. Eksempler Bildene viser GFP immunoreaktivitets i (C) retikulære thalamus og globus pallidus, (D) dentate gyrus og (E) i CA1 regionen i hippocampus. DG, dentate gyrus, RT, retikulære thalamic kjernen; GP, globus pallidus. Scale barer: B, 500 mikrometer, C, 100 mikrometer, D, 100 mikrometer, E, 500 mikrometer.

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rAAV vektorer blir stadig mer verdifull for in vivo studier på dyr. Her har vi beskrevet en enkel og rimelig protokoll for å produsere rAAVs, noe som kan lette den utbredte anvendelsen av denne nyttige vektor ved å unngå kostbar outsourcing av virus produksjonen til bedrifter. Protokollen (basert på referanse 1) beskriver produksjonen av kimære rAAV1 / 2 vektorer som inneholder kapsid proteiner fra begge foreldrenes serotyper på lik forholdstall fire. Rensing av rAAV vektorer ved binding til heparin kolonner avhengig uttrykk for AAV2 kapsid proteiner, men kan kombineres med serotyper enn AAV1 skissert her. I tillegg har AAV6 blitt rapportert å binde seg til heparin, men med redusert affinitet, og kan potensielt bli renset med heparin kolonner ved hjelp av denne prosedyren 5,6. Det bør bemerkes imidlertid at andre serotyper vil kreve ulike konsentrasjoner av NaCl for heparin kolonnen eluering 5,7. Emballasje rAAV genomer ble vist å være optimal mellom 4,1 og 4,9 kb med en kraftig reduksjon i emballasje effektivitet opp til 5,2 kb 8. Denne emballasjen grensen kan betraktes som den største ulempen av rAAV system, siden celletype-spesifikk regulering av transgene uttrykket oppnås vanligvis ved store cis handle elementer som ikke kan innpasses innenfor den lille AAV partikler. For å overvinne lavt titer partier, slik som de som følge av forsøk på å pakke gener som er nær AAV pakking grense vi kombinere separate viral partier konsentrert til 250 mL i en 500 mL batch, snarere enn å legge PBS som beskrevet i punkt 6.3. Pålitelig celle-type spesifikke transduksjon kan oppnås ved å kombinere Cre-driver mus og betinget rAAV kassetter (se nedenfor) for å unngå strekk emballasjen grensen. Av notatet, mens denne protokollen forsøker å gi enkle å følge instruksjonene på produksjon av høy tITER rAAV, det krever tilstedeværelse av AAV2 kapsid protein moieties for rensing av heparin affinitet kromatografi. Serotyper enn AAV2 må bli renset ved hjelp av alternative prosedyrer 9. En fordel med heparin kolonne rensing er det antas å produsere AAV vektorer som har større smittsomhet enn dem som produseres ved hjelp av en cesium klorid gradient 10. Men det er også noen ulemper med denne metoden. For eksempel kan andre proteiner som binder heparin også være til stede i renset viral lager. Mens tropisme av heparin-renset vektorer kan bli påvirket av vektor 10 titer, vår tilnærming spesifikt mot enten eksitatoriske 20 eller inhibitoriske nevroner (fig 2) sysselsetter Cre-indusert aktivering av AAV-mediert transgenet uttrykk og er titre-uavhengig. Et alternativ til heparin kolonne rensing er bruken av en iodixanol tetthet gradient, noe som gir en høyere viral titer og renere forberedelse enn bruk aven cesium klorid gradient 10. Denne metoden kan også kombineres med heparin kolonne renselse å produsere en vektor som er større enn 99% ren 11. Derfor må nøye overveielse til å bli tatt av enkelte grupper om hvilke viral rensing metoden er best for deres spesielle nedstrøms søknaden. Det vanligste problemet forbundet med denne protokollen er lav viral titer. Vår erfaring er dette kan vanligvis spores til lav transfeksjon effektivitet, eller eluering av virus fra heparin kolonnene. Alle transfeksjon materialer bør ha romtemperatur før transfeksjon, og test transfections bør utføres for å finne optimale forhold i hvert enkelt laboratorium. Andre metoder for transfeksjon, som lipofectamine, er svært effektiv, men kan være uoverkommelig dyrt. I tillegg blir konsentrasjonen og pH i heparin kolonnen elueringsmiddel løsninger kritisk for komplett eluering av virioner fra heparin kolonner withoUT skade. Et annet viktig poeng er at emballasjen cellene må følge godt til overflaten av vev kultur retter. Hvis cellene løsner på et tidspunkt under prosedyren er det anbefalt å avslutte forsøket og avriming en ny hetteglass av celler. Spatio-temporal kontroll av transgene uttrykk hos gnagere er lett oppnås ved nøyaktige anatomiske målretting og det utviklingsstadiet av dyret. Effektiv AAV-mediert genoverføring har vært vist i utero 12,13. I den voksne hjernen, kan området infisert med rAAV partikler etter stereotaxic injeksjon justeres fra svært små målrettede regioner, til mye større områder, ikke bare av endringer i viral titer, men også ved å endre injeksjon parametere. Disse inkluderer volum og hastighet av injeksjoner og inkludering av polyol mannitol med viruset suspensjon 14. Mannitol kan også brukes til å forbedre infeksjon av en rekke vev etter systemisk rAAV injeksjon 15 &lt;/ Sup&gt;. Emballasjen kapasitet rAAV (ca 4,7 kb) er trolig den mest begrensende faktoren i forhold til gener som kan uttrykkes fra disse virale vektorer. Imidlertid har nyere studier vist at dette kan være delvis overvinnes ved å splitte større gener eller uttrykk kassetter i separate rAAV vektorer og innføre en bridging sekvens, initiere genekspresjon når virioner infiserer samme celle 16. Expression nivåer av gener båret av rAAV vektorer kan også bli sterkt forbedret ved hjelp av self-gratis rAAV vektorer 17. Stereotaxic injeksjon av rAAV vektorer gir en rask, billig og kraftig metode for å indusere genuttrykk i en region-spesifikk måte. I kombinasjon med 2. generasjons RNA-interferens strategier 18 rAAVs kan også brukes for region-spesifikke gen-knock ned. rAAVs kan kombineres med Cre-transgene mus eller celle-typespesifikke arrangører å oppnå krets-og celle-type-Spesifikke genuttrykk, tillater det genetiske manipulasjoner med høy romlig oppløsning 2,19-21, mens montering rAAVs med f.eks Tet-systemet legger temporal kontroll over virus-mediert genuttrykk 22,23. Disse eksemplene illustrerer det enorme kombinatorisk potensialet i disse vektorer og spår en rask økning i rAAV-baserte studier i årene som kommer.

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Amicon Ultra sentrifugal filtre </td><td> Millipore </td><td> UFC810024 </td><td> 100000 MWCO </td></tr><tr><td> Benzonase nukleasefritt </td><td> Sigma-Aldrich </td><td> E1014 </td><td></td></tr><tr><td> Dulbeco modifiserte Eagle medium (DMEM) </td><td> Invitrogen </td><td> 10567014 </td><td> Suppler med 10% FCS og 10 enheter / ml penicillin/100 mikrogram / ml streptomycin </td></tr><tr><td> Hek293 celler </td><td> Amerikansk type kultur samling </td><td> CRL-1573 </td><td> Bruk på passasjen mindre enn 30 </td></tr><tr><td> HEPES buffered saltvann </td><td> Sigma-Aldrich </td><td> 51558 </td><td></td></tr><tr><td> HiTrap Heparin kassett </td><td> Sigma-Aldrich </td><td> 54836 </td><td></td></tr><tr><td> Iscove modifiserte Dulbecco medium </td><td> Invitrogen </td><td> 12440-053 </td><td> Suppler med 5% FCS </td></tr><tr><td> Sodium deoxycholate </td><td> Sigma-Aldrich </td><td> D5670 </td><td> Lag friske løsning for hver batch </td></tr><tr><td> 15 cm Tissue kultur retter </td><td> Fisher Scientific </td><td> 157150 </td><td></td></tr><tr><td> Stbl2 kompetente celler </td><td> Invitrogen </td><td> 10268-019 </td><td></td></tr><tr><td> Virkon-løsning </td><td> Fisher Scientific </td><td> NC9821357 </td><td> La desinfisere over natten før du kasserer for å drenere </td></tr></tbody></table> Tabell 1. Spesifikke reagenser som kreves for protokoll

production and titering of recombinant adeno-associated viral vectors

I de senere årene rekombinant adeno-assosiert virale vektorer (AAV) har blitt stadig mer verdifull for in vivo studier i dyr, og blir også testet i kliniske studier. Wild-type AAV er en ikke-patogene medlem av parvoviridae familien og iboende replikering-mangelfull. Den brede transduksjon profil, lavt immunforsvar, samt den sterke og vedvarende transgenet uttrykk oppnådd med disse vektorene har gjort dem en populær og allsidig verktøy for in vitro og in vivo genet levering. rAAVs kan enkelt og billig produsert i laboratoriet, og basert på deres gunstig sikkerhetsprofil, er generelt gitt en lav sikkerhet klassifisering. Her beskriver vi en metode for produksjon og titering av kimære rAAVs inneholder kapsid proteiner både AAV1 og AAV2. Bruken av disse såkalte kimære vektorer kombinerer fordelene av begge foreldrenes serotyper som høye titre aksjer (AAV1) og rensingav affinitet kromatografi (AAV2). Disse AAV serotyper er best studert av alle AAV serotype og individuelt har en bred smittsomhet mønster. Den kimære vektorer beskrevet her bør ha smittsomme egenskaper AAV1 og AAV2 og kan dermed forventes å infisere et stort spekter av vev, inkludert nerveceller, skjelettmuskulatur, bukspyttkjertel, nyre blant andre. Metoden som beskrives her bruker heparin kolonne renselse, trodde en metode for å gi en høyere viral titer og renere viral forberedelse enn andre rensemetoder, slik som sentrifugering gjennom et cesium klorid gradient. I tillegg beskriver vi hvordan disse vektorene kan raskt og enkelt titered å gi nøyaktig lesing av antall infeksiøse partikler produsert.

Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Production and Titering of Recombinant Adeno-associated Viral Vectors

Rekombinant adeno-assosiert virus (rAAVs) vektorene blir stadig mer verdifull for<em&gt; In vivo</em&gt; Studier hos dyr. Vi beskriver hvordan rAAVs kan produseres i laboratoriet og hvordan disse vektorene kan titered å gi en nøyaktig avlesning av antall infeksiøse partikler produsert.

Produksjon og Titering av rekombinant Adeno-assosiert virale vektorer

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

production-and-titering-of-recombinant-adeno-associated-viral-vectors

Research

JoVE Journal

Immunology and Infection

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Effektiv Rekombinant Parvovirus Produksjonen ved hjelp av Adenovirus deriverte Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Engineering and Evolution of Synthetic Adeno-assosiert virus (AAV) genterapi vektorer via DNA Family shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Produksjon og rensing av Non replicative Canine adenovirus type 2 Avledet Vektorer

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissekere medfødte immunsignalisering i Viral Evasion av cytokinproduksion

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Utvikling av lyofilisert Loop-mediert Isotermiske amplifiseringsreagenser for deteksjon av<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Måling av Influensa neuraminidase Inhibering antistofftitere ved enzym-bundet lektin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Pinne Prøvetaking metode for påvisning av humant Norovirus på overflater

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...