Name: production and titering of recombinant adeno-associated viral vectors
Uploaded: 2011-11-27T14:00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Se figur 1 for en illustration opsummerer de følgende protokol. Sikkerhed Bemærk: Alt materiale, der har været i kontakt med samlet viruspartikler skal desinficeres med Virkon løsning eller andet egnet desinfektionsmiddel. 1. Udarbejdelse af plasmid DNA bestande (~ 2 dage) <ol><li> Følgende plasmider kræves 1: <ul><li> pRV1 - Med indhold af AAV2 Rep og Cap sekvenser </li><li> pH21 - Med indhold af AAV1 Rep og Cap sekvenser </li><li> pFdelta6 - Adenovirus-hjælper plasmid </li><li> AAV plasmid, som indeholder den rekombinante udtryk kassetten flankeret af AAV2 emballage signaler (omvendt terminal gentager, ITRS) </li></ul></li><li> Mens pFdelta6 skal dyrkes i Stbl2 kompetente celler for at undgå delvis sletning, kan pRV1 og pH21 dyrkes i DH5alpha kompetente celler. AAV plasmider kan dyrkes i Stbl2 celler, hvis delvise sletninger forekomme i DH5alpha celler. </li><li>Plasmid DNA skal være af høj kvalitet og fri for RNA forurenende stoffer. Plasmider kan screenes for integritet ved hjælp af følgende fordøjer: <ul><li> pRV1 - Digest med XbaI at give bands på 7,5 kb og 3,8 kb </li><li> pH21 - Digest med EcoRI at give bands på 4,5 kb, 2,8 kb og 0,2 kb </li><li> pFdelta6 - Digest med HindIII at give bands på 5,5 kb, 3 KB, 3 kb, 2,3 kb og 1,5 kb </li></ul></li></ol> 2. Udarbejdelse af humane embryonale Nyre 293 (Hek293) celler til transfektion (2 - 3 dage) <ol><li> Plate to 80% sammenflydende 150 cm 2 flasker af Hek293 celler i fem 15 cm diameter Nunc vævskultur retter. Cellerne skal være 70 - 80% sammenflydende før transfektion (ca. 48 timer efter plating). Kultur celler i standard Dulbecco ændrede Eagle medium (DMEM) med lav glukose indeholder 10% føtalt kalveserum og 100 E / ml penicillin / 100 mg / ml streptomycin. </li><li> 3 timer før transfektion fjerner DMEM og erstatte med Iscove&#39;S modificeret Dulbecco er medium (IMDM) indeholdende 5% føtalt kalveserum. </li></ol> 3. Transfektion af virale plasmider (~ 1 time til transfektion, 3 dage for inkubation) <ol><li> Forbered følgende for et enkelt parti (5 x 15 cm vævskultur plader) af 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 μl 2,5 M CaCl 2 </li><li> 12 ml dH 2 O </li></ul></li><li> I en klasse 2 vævskultur hætte sterile filtrere transfektion blandingen i et 50 ml rør. </li><li> Mens hvirvelblanding løsningen, hurtigt at tilføje 13 ml 2 x HEPES bufferet saltvand (pH 7,05). Sæt låg på 50 ml rør og fortsætter med at vortex i 15 sekunder. Lad dem stå i 1 minut 45 sekunder, skal et fint hvidt bundfald form. </li><li> Forsigtigt tilsættes 5 ml af transfektion løsningen på hver 15 cm vævskultur fad. Swirl plader at blande og vende tilbage til kuvøsen. </li><li> 16 timer efter transfektion fjerne IMDM medium og erstatte med DMEM. </li></ol> 4. Lysing af celler og høst af rAAVs (2 timer) <ol><li> 72 timer efter transfektion, fjerne medier fra cellekultur plader og kassér. Alt affald skal behandles med Virkon løsning eller andet egnet desinfektionsmiddel. </li><li> Vask forsigtigt cellerne i varme 1x fosfatbufferet saltopløsning (PBS, pH 7,4). </li><li> Tilsæt 25 ml varm PBS til hver plade, og forsigtigt fjerne celler med en celle skraber. Saml suspension i 50 ml rør. </li><li> Pellet cellerne ved 800 xgi 10 minutter. </li><li> Kassér supernatanten og resuspender pellet i 150 mM NaCl, 20 mM Tris pH 8,0, bruge 10 ml pr vævskultur plade. Opdelt i to 50 ml rør. </li><li> Forbered en frisk opløsning af 10% natriumdeoxycholat i DH 2 O. Tilsæt 1,25 ml af denne til hvert rør i en endelig koncentration på 0,5%. Tilføj benzonase nuklease til en endelig koncentration på 50 enheder per ml. Bland rør grundigt. </li><li&gt; inkuberes ved 37 ° C i 1 time. </li><li> Fjern celleaffald ved centrifugering ved 3000 xgi 15 min. Overførsel til frisk 50 ml tube, sikre, at alle cellefragment er blevet fjernet for at forhindre blokering af heparin kolonner (se trin 5). På dette stadium, kan prøverne opbevares ved -20 ° C, før du fortsætter. Vi har gemt prøver i flere uger ved -20 ° C uden en reduktion i smitteevne. </li></ol> 5. Heparin kolonne oprensning af rAAVs (2 - 3 timer) <ol><li> Opsætning HiTrap heparin kolonner ved hjælp af en peristaltisk pumpe, så løsninger flow gennem kolonnen med 1 ml per minut for trin fra 5,2 til 5,4. Det er vigtigt at sikre, at ingen luftbobler er indført i heparin kolonnen. </li><li> Ligevægt kolonnen med 10 ml 150 mM NaCl, 20 mM Tris, pH 8,0. </li><li> Anvend 50 ml virus løsning på kolonnen og lad dem flyde igennem. </li><li> Vask kolonnen med 20 ml 100 mM NaCl, 20 mM Tris, pH 8,0. </li><li> Ved hjælp af en 5 ml sprøjte fortsætte med at vaske kolonnen wed 1 ml 200 mM NaCl, 20 mM Tris, pH 8,0, efterfulgt af 1 ml 300 mM NaCl, 20 mM Tris, pH 8,0. Kassér gennemstrømning. </li><li> Brug 5 ml sprøjter og blide tryk elueres virus fra kolonnen ved at anvende: <ul><li> 1,5 ml 400 mM NaCl, 20 mM Tris, pH 8,0 </li><li> 3,0 ml 450 mM NaCl, 20 mM Tris, pH 8,0 </li><li> 1,5 ml 500 mM NaCl, 20 mM Tris, pH 8,0 </li><li> Saml disse i et 15 ml centrifugerør. </li></ul></li></ol> 6. Koncentration og steril filtrering af rAAVs (1 time) <ol><li> Koncentrat vektor hjælp Amicon ultra-4 centrifugal filteranlæg med en 100.000 molekylvægt cutoff. Load 4 ml af kolonne eluat i koncentrator og centrifuger ved 2000 xgi 2 minutter (ved stuetemperatur). Kassér gennemstrømning og genindlæs koncentrator med resterende virus løsning, og gentag centrifugering. Den koncentrerede mængde bør være cirka 250 μl. Hvis koncentrerede volumen er betydeligt mere end dette, kasseres flow through og fortsætte med at centrifugere i et minut trin, indtil lydstyrken er cirka 250 μl. </li><li> Tilsæt 250 μl af PBS til at virus til et endeligt volumen på 500 μl og fjerne fra koncentrator. </li><li> Filtrer vektor gennem et 13 mm diameter 0,2 μm sprøjte filter. Vector skal alikvoteres og opbevares ved -80 ° C, indtil påkrævet. </li></ol> 7. Titering af virale lagre (3 dage) Det kræver en promotor, der er aktiv i Hek293 celler køre en reporter gen eller en immunocytochemically påviselig gen produkt. Ved fremstilling af en vektor, der ikke er kompatibel med Hek293 celler andre cellelinjer og primære cellekulturer kan anvendes. <ol><li> Forbered 18 poly-L-lysin-belagte glas dækglas eller 18 brønde i Nunc kammer dias ved såning Hek293 celler, så de er 40 - 50% sammenflydende. Ved titrering af Cre-afhængige rAAVs brug Hek293 celler, der stabilt udtrykker Cre recombinase 2. </li><li> Inficere hver brønd med seriel dilutions af AAV vektor (Fig. 2A). Brug 3 brønde pr fortynding. Vi rutinemæssigt tilføjer virus direkte til hver brønd. </li><li> Efter 72 timer fix Hek293 celler ved at tilføje en tilsvarende mængde på 4% paraformaldehyd (PFA) til medium (hvilket giver en endelig koncentration på 2% PFA) i ti minutter ved stuetemperatur. </li><li> Vask celle tre gange i PBS, skylles i dH 2 O og dækglas. </li><li> Tæl antallet af transduced celler fra de tre brønde, der har den højeste fortyndingsfaktoren, men stadig indeholder inficerede celler, finde gennemsnittet af disse og ganges med fortyndingsfaktoren for at oplyse om antallet af infektiøse enheder pr mikroliter (Fig. 2B). </li></ol> 8. Forventede resultater Hek293 celler transduced med en virus kodning forbedret grønt fluorescerende protein (eGFP) er vist i figur 2B. Vi generelt opnå konsistente titre af omkring 6 x 10 6 infektiøse partikler per mikroliter. Vi rutinemæssigt bruger disse vektorer for stereotaxic injeIndsatsen i den voksne gnavere hjernen. Dette giver en kraftfuld teknik til region-specifikke genekspression 2. At kombinere denne region specificitet med celle-typen selektive genekspression vi injicere Cre recombinase-afhængige rAAVs i mål-regioner Cre-transgene mus 2. Figur 2C-E viser eksempler på stereotaxic injektioner af Cre-afhængige rAAVs kodning eGFP i forskellige områder i hjernen af voksne parvalbumin-Cre transgene mus 3. <img alt="Figur 1" src="/files/ftp_upload/3348/3348fig1.jpg" /> Figur 1. Skematisk illustration af protokollen for rAAV produktion. . 1) Hek293 celler er belagt og er vokset til 70 - 80% sammenløb. 2). AAV og hjælper plasmider er forberedt og transficeret ind Hek293 celler. 3.) Medium er ændret tilbage til DMEM 16 timer efter transfektering og Hek293 celler dyrkes i endnu 56 timer. 4). Hek293 celler er høstet og lyseret. rAAV vektorer er adskilt fra cellefragment af centrifugation. 5). Viral partikler er renset gennem heparin kolonner, før koncentrationen og sterilisering. 6.) Hek293 celler dyrkes den 24 samt plader og der inficeres med serielle fortyndinger af AAV vektor til at bestemme antallet af infektiøse enheder. <img alt="Figur 2" src="/files/ftp_upload/3348/3348fig2.jpg" /> Figur 2. Titering og in vivo anvendelse af rAAV1 / 2. (A) Opsætning af Hek293 celler til måling af viral titer. Hek293 celler er transduced med serielle fortyndinger af rAAVs. C, uinficerede kontrol, N, 1 ml af ufortyndet viral lager. (B) Hek293 celler inficeret med rAAVs udtrykke eGFP. (CE) Viral eGFP udtryk er begrænset til Cre-udtrykkende neuroner i forskellige mål regioner parvalbumin-Cre transgene mus efter stereotaxic injektion af Cre-aktiverede rAAVs. Eksempler billeder viser GFP immunoreaktivitet i (C) reticular thalamus og globus pallidus, (D) dentate gyrus og (E) CA1 region i hippocampus. GD, dentate gyrus, RT, retikulære thalamic kerne; GP, globus pallidus. Skala barer: B, 500 μm, C, 100 mM, D, 100 mM, E, 500 μm.

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rAAV vektorer bliver mere og mere værdifulde for in vivo undersøgelser på dyr. Her har vi beskrevet en simpel og billig protokol til fremstilling rAAVs, der kan lette den udbredte anvendelse af denne nyttige vektor ved at undgå de dyre outsourcing af virus produktion til virksomheder. Protokollen (baseret på reference 1) beskriver produktion af kimære rAAV1 / 2 vektorer indeholdende kapsid proteiner fra begge forældrenes serotyper med lige nøgletal 4. Rensning af rAAV vektorer ved binding til heparin kolonner afhængig af ekspressionen af ​​AAV2 kapsid proteiner, men kan kombineres med serotyper end AAV1 skitseret her. Derudover har AAV6 blevet rapporteret at binde sig til heparin, dog med en reduceret affinitet, og potentielt kunne renses med heparin kolonner ved hjælp af denne procedure 5,6. Det skal dog bemærkes, at andre serotyper vil kræve forskellige koncentrationer af NaCl for heparin kolonne eluering 5,7. Emballage rAAV genomer viste sig at være optimalt mellem 4,1 og 4,9 kb med en kraftig reduktion i emballage effektivitet på op til 5,2 KB 8. Denne emballage grænse kan betragtes som den største ulempe ved rAAV systemet, da celletype-specifikke regulering af transgene udtryk normalt opnås ved store cis handler elementer, som ikke kan rummes inden for de små AAV partikler. For at overvinde lav titer partier, såsom dem, der skyldes at forsøge at pakke gener, der er tæt på AAV pakning grænsen vi kombinerer separate viral partier koncentreret til 250 μl i en 500 μl parti, snarere end at tilføje PBS som beskrevet i trin 6.3. Pålidelig celle-typespecifikke transduktion kan opnås ved at kombinere Cre-driver mus og betingede rAAV kassetter (se nedenfor) for at undgå at strække emballagen grænse. Det skal bemærkes, mens denne protokol forsøger at give let at følge instruktionerne på produktion af høj titer rAAV, det kræver tilstedeværelse af AAV2 kapsid protein fragmenter for rensning ved heparin affinitetskromatografi. Serotyper end AAV2 skal renses ved hjælp af alternative procedurer 9. En fordel ved heparin kolonne rensning er det menes at producere AAV vektorer, som har større infektivitet end produceret dem der bruger en caesiumchlorid gradient 10. Der er dog også nogle ulemper ved denne metode. For eksempel kan andre proteiner, der binder heparin også være til stede i den rensede virus-bestanden. Mens tropisme af heparin-renset vektorer kan være påvirket af vektoren titer 10, vores tilgang til specifikt at målrette enten excitatoriske 20 eller hæmmende neuroner (fig. 2) beskæftiger Cre-induceret aktivering af AAV-medieret transgene udtryk og er titer-uafhængig. Et alternativ til heparin kolonne rensning er brugen af ​​en iodixanol densitetsgradient, som producerer en højere viral titer og renere forberedelse end brugen afen caesiumchlorid gradient 10. Denne metode kan også kombineres med heparin kolonne rensning for at producere en vektor, der er større end 99% ren 11. Derfor omhyggelige overvejelser skal tages af de enkelte grupper, som viral rensning metode er bedst for deres særlige downstream anvendelse. Det mest almindelige problem forbundet med denne protokol er lavt viral titer. Det er vores erfaring dette kan som regel spores tilbage til lav transfektion effektivitet, eller eluering af virus fra heparin kolonner. Alle transfektion materialer bør have stuetemperatur før transfektion, og test transfections skal udføres for at finde de optimale betingelser i hvert enkelt laboratorium. Andre metoder til transfektion, såsom lipofectamine, er meget effektive, men det kan være uoverkommeligt dyre. Derudover koncentrationen og pH af heparin kolonnen elueringen løsninger er afgørende for komplet eluering af virioner fra heparin kolonner without skader. Et andet vigtigt punkt er, at emballagen celler skal holde godt på overfladen af ​​vævskultur retter. Hvis cellerne løsne sig på et tidspunkt i løbet af proceduren anbefales det at afslutte eksperimentet og afrimning et nyt hætteglas med celler. Spatio-temporale kontrol af transgene udtryk i gnavere er let opnås ved at præcise anatomiske målrette og udviklingsstadium af dyret. Effektiv AAV-medieret genoverførsel er blevet vist i livmoderen 12,13. I den voksne hjerne, kan området inficeret med rAAV partikler efter stereotaxic injektion justeres fra meget små målrettede regioner, til langt større områder, der ikke kun af ændringer i den virale titer, men også ved at ændre injektion parametre. Disse indbefatter bl.a. mængde og hastighed af injektioner og inddragelsen af polyol mannitol med virussuspension 14. Mannitol kan også bruges til at øge infektion i en række forskellige væv efter systemisk rAAV injektion 15 &lt;/ Sup&gt;. Emballagen kapacitet rAAV (cirka 4,7 kb) er nok den mest begrænsende faktor i forhold til de gener, der kan udtrykkes fra disse virale vektorer. Imidlertid har nyere undersøgelser vist, at dette delvis kan overvindes ved at dele større gener eller udtryk kassetter i separate rAAV vektorer og indføre en bro sekvens, indleder genekspression, når virioner inficere den samme celle 16. Expression niveauer af gener bæres af rAAV vektorer kan også blive øget væsentligt ved hjælp af selv-gratis rAAV vektorer 17. Stereotaxic injektion af rAAV vektorer giver en hurtig, billig og effektiv metode til at fremkalde genekspression i en region-specifik måde. I kombination med 2. generation RNA-interferens strategier 18 rAAVs kan også bruges til region-specifikke gen-knock down. rAAVs kan kombineres med Cre-transgene mus eller celle-typespecifikke initiativtagere at opnå kredsløbs-og celle-type-Specifik genekspression, som gør det genetiske manipulationer med høj rumlig opløsning 2,19-21, mens montering rAAVs med fx Tet-systemet tilføjer tidsmæssig kontrol over virus-medieret genekspression 22,23. Disse eksempler illustrerer den enorme kombinatoriske potentiale i disse vektorer og forudsige en hurtig stigning i rAAV-baserede undersøgelser i løbet af de kommende år.

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Amicon Ultra centrifugal filtre </td><td> Millipore </td><td> UFC810024 </td><td> 100.000 MWCO </td></tr><tr><td> Benzonase nuklease </td><td> Sigma-Aldrich </td><td> E1014 </td><td></td></tr><tr><td> Dulbeco ændrede Eagle medium (DMEM) </td><td> Invitrogen </td><td> 10567014 </td><td> Supplere med 10% FCS og 10 enheder / ml penicillin/100 mg / ml streptomycin </td></tr><tr><td> Hek293 celler </td><td> Amerikansk type kultur samling </td><td> CRL-1573 </td><td> Brug ved passage mindre end 30 </td></tr><tr><td> HEPES bufferet saltvand </td><td> Sigma-Aldrich </td><td> 51558 </td><td></td></tr><tr><td> HiTrap Heparin patron </td><td> Sigma-Aldrich </td><td> 54836 </td><td></td></tr><tr><td> Iscove ændrede Dulbecco mellemfristede </td><td> Invitrogen </td><td> 12440-053 </td><td> Supplere med 5% FCS </td></tr><tr><td> Natriumdeoxycholat </td><td> Sigma-Aldrich </td><td> D5670 </td><td> Gør frisk løsning for hvert enkelt parti </td></tr><tr><td> 15 cm Vævskultur 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> Lad at desinficere natten før de kasserer til at dræne </td></tr></tbody></table> Tabel 1. Specifikke reagenser, der kræves til protokol

production and titering of recombinant adeno-associated viral vectors

I de senere år rekombinant adeno-associeret virale vektorer (AAV) er blevet stadig mere værdifuld for in vivo undersøgelser på dyr, og er også i øjeblikket testet i humane kliniske undersøgelser. Vildtype-AAV er en ikke-patogene medlem af parvoviridae familien og i sagens natur replikationskompetente mangelfuld. Den brede transduktion profil, lavt immunforsvar samt den stærke og vedvarende transgenet udtryk opnås med disse vektorer har gjort dem til et populært og alsidigt værktøj for in vitro og in vivo gen levering. rAAVs kan nemt og billigt produceret i laboratoriet, og baseret på deres gunstige sikkerhedsprofil, er generelt får en lav sikkerhed klassifikation. Her beskriver vi en metode til produktion og titering af kimære rAAVs indeholder kapsid proteiner af både AAV1 og AAV2. Brugen af ​​disse såkaldte kimære vektorer kombinerer fordelene af begge forældres serotyper såsom høje titre bestande (AAV1) og rensningved affinitets kromatografi (AAV2). Disse AAV serotyper er de bedst undersøgte af alle AAV serotyper, og individuelt har en bred infektivitet mønster. De kimære vektorer, der er beskrevet her, bør have smitsomme egenskaber AAV1 og AAV2 og kan således forventes at inficere en lang række væv, herunder neuroner, skeletmuskulatur, bugspytkirtel, nyre blandt andre. Den metode, der beskrives her bruger heparin kolonne oprensning, en metode som menes at give en højere viral titer og renere virale forberedelser end andre rensning metoder, såsom centrifugering gennem en caesiumchlorid gradient. Derudover beskriver vi, hvordan disse vektorer kan hurtigt og nemt titered at give nøjagtig måling af antallet af smitsomme producerede partikler.

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-associeret virus (rAAVs) vektorer bliver stadig mere værdifuld for<em&gt; In vivo</em&gt; Studier i dyr. Vi beskriver, hvordan rAAVs kan fremstilles i laboratoriet, og hvordan disse vektorer kan titered at give en præcis aflæsning af antallet af smitsomme producerede partikler.

Produktion og Titering af rekombinant adeno-associeret 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 ...

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 Produktion ved hjælp af adenovirusafledte 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 af syntetisk Adeno-associeret virus (AAV) Genterapivektorer via DNA Family blander

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.

Produktion og oprensning af ikke replikativ Canine adenovirus type 2-afledte 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 immunforsvar signalering i Viral Evasion af cytokinproduktion

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.

Udviklingen af ​​Frysetørret Loop-medierede Isotermiske Amplification reagenser til påvisning af<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 Influenza neuraminidase Inhibering Antistoftitere af Enzyme-linked lectin 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.

Swab Sampling metode til påvisning af menneskelige Norovirus på overflader

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

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.