Ourhighly-efficient3rdgenerationlentiviruspackagingmixreagentcontaininganoptimisedplasmidsetatcarefully-definedratiosformaximumvectorproduction
ProductDetails:
HighTitrePackagingMixesandPlasmidSystemsAvailableDesignedto:
•MinimiseRecombination–UsingUniquePromoters,UTRsandPoly-adenylationSignals
•MaximiseProteinExpressionUsingProprietaryDNADesignAlgorithms
•MaximiseTitres–ComparedwithLifeTech,ClonTech,SigmaandOtherCommercialSystems
•MaximiseTransfectionEfficiencyByMinimisingPlasmidSize
•ProvidetheMostCostEffectiveSolutionforLargeScaleProduction
Thisreagentisdesignedtobeco-transfectedintoHEK-293orHEK-293Tcellswithaplasmidcontaininglentivirusgenomeencodingageneofinterest.High-titrevirussupernatentscanthenharvestedandusedtoinfectarangeofcelltypes.ThispackagingsystemcontainsanoptimisedformoftheVSVGglycoproteinwhichprovidebroadinfectioustropisminmanycelltypes.Otherglycoproteinvariantsareavailableonrequest.
LentivirusData
Following2yearsofintensivedevelopment,OxfordGeneticsDNAdesignengineershavedevelopedthenextgenerationoflentivirusexpressionsolutions(SnapFastProLTX).UsingtheindustryleadingSnapFastTMcloningplatformtheyscreened>5000recombinantpromoters,UTRsandpolyAsignalstocreateexpressioncassettesthatminimisethechancesofrecombinationandmaximisevirusproduction.
PackagingMixes:
•Halfthecostperplatecomparedwithallothercompetingsystems
•Optimisedconcentrationsandratiosofthecomponentplasmids
•Availableinsmallandlargequantitiesforpilotstudiesandlargescalemanufacture
•Detailedprotocolsavailableonrequest
Protocol:
TransfectionofHEK293cellswithLentiviralDNAtoproducelentivirusina10cmplate
1.Eighteentotwenty-fourhourspriortotransfection,seed1-2x106cells/mlHEK293cells(HEK293Tcellscanalsobeusedandmayprovideashighervirustitre)perwellinaPoly-L-Lysine10cmplatein15mlofHighGlucoseDulbecco’sModifiedEagleMedium(DMEM)with10%FetalBovineSerum(FBS).
•Swirlingofthewellshouldbeavoidedtopreventcellsclumpinginthecentre.
•Afterincubationat37˚Cfor18-24hours,wellsarecheckedvisuallytohave~70-80%confluency(cellsat100%confluencyshouldnotbeusedasthiswillreducetransfectionefficiency).
2.Foreach10cmdish:
•TubeA:Intoa1.5mlpolypropylenetubeadd150µlofSnapFastProLTX packagingmixand6.25µgofyourlentiviralvectorgenomeplasmid.Makethetotaltubevolumeupto1.1mlwithOpti-MEM®(ThermoFisherScientific®)andthoroughlymixbypipettingupanddown.
•TubeB:Intoa1.5mlpolypropylenetubeadd55µlof25kDabranchedpolyethylenimine(PEI)(Sigma-Aldrich®)(stockconcentration1mg/ml)and1.1mlofOpti-MEM®.
•TubeC:Mix1.05mlfrombothtubeAandBintoa15mlpolypropyleneFalcontube.InvertthetubemultipletimestoensuretheDNA:PEIismixed.Avoidvortexingorvigorouspipetting.AllowtheDNA:PEItocomplexatroomtemperaturefor20minutes.
3.Beforetransfection,replacethetissueculturemediaofeachwelltobetransfectedwith8mlofDMEM(10%FBS)andplacebackintheincubatorfortenminutestoallowthetemperaturetorecover.
4.AftertubeChascomplexed,add2mlinadropwisefashiontothewell.
5.IncubatetheplateinahumidifiedCO2incubatorat37˚Cfor72hours.
6.Collectthesupernatantintoapoly-propylenetubeandspinat5000gfor2minutespelletanycellsthathavedislodgedfromtheplateduringvirusproduction.Harvestthesupernatantandavoiddisturbinganycellsthatmaybeatthebottomofthetube.
TransfectionofHEK293cellswithLentiviralDNAtoproducelentivirusina6-wellplate
1.Eighteentotwenty-fourhourspriortotransfection,seed1x105cells/mlHEK293cells(HEK293Tcellscanalsobeusedandmayprovideashighervirustitre)perwellinaPoly-L-Lysine6-WellPlatein3mlofHighGlucoseDulbecco’sModifiedEagleMedium(DMEM)with10%FetalBovineSerum(FBS).
•Swirlingofthewellshouldbeavoidedtopreventcellsclumpinginthecentre.
•Afterincubationat37˚Cfor18-24hours,wellsarecheckedvisuallytohave~70-80%confluency(cellsat100%confluencyshouldnotbeusedasthiswillreducetransfectionefficiency).
2.Foreachsinglewellofa6-wellplate:
•TubeA:Intoa1.5mlpolypropylenetubeadd30µlofSnapFastProLTXpackagingmixand1.25µgofyourlentiviralvectorgenomeplasmid.Makethetotaltubevolumeupto220µlwithOpti-MEM®(ThermoFisherScientific®)andthoroughlymixbypipettingupanddown.
•TubeB:Intoa1.5mlpolypropylenetubeadd11µlof25kDabranchedpolyethylenimine(PEI)(Sigma-Aldrich®)(stockconcentration1mg/ml)and209µlofOpti-MEM®.
•TubeC:Mix210µlfrombothtubeAandB.InvertthetubemultipletimestoensuretheDNA:PEIismixed.Avoidvortexingorvigorouspipetting.AllowtheDNA:PEItocomplexatroomtemperaturefor20minutes.
3.Beforetransfection,replacethetissueculutremediaofeachwelltobetransfectedwith2mlofDMEM(10%FBS)andplacebackintheincubatorfortenminutestoallowthetemperaturetorecover.
4.AftertubeChascomplexed,add400µlinadropwisefashiontothewell.
5.IncubatetheplateinahumidifiedCO2incubatorat37˚Cfor72hours.
6.Collectthesupernatantintoapoly-propylenetubeandspinat5000gfor2minutespelletanycellsthathavedislodgedfromtheplateduringvirusproduction.Harvestthesupernatantandavoiddisturbinganycellsthatmaybeatthebottomofthetube.
Notes:
Thesupernatantcanbestoredforuptoaweekat4˚Corindefinitelyat-80˚C.
Usingothertransfectionreagentscansignificantlyimproveyieldsbutwillincreasethecostofvectorproductionsignificantly.
Usepoly-propylenewherepossIBLeandavoidusingpolystyrenetubeswherepossiblebecausethesurfacescancarryachargewhichcancausevirusestostick.
MeasurementofTitrebyInfectionofHEK293cellswithLentivirusProducedbyTransfectionina48-WellPlate.
1.Twenty-fourhourspriortovirusinfection,seed3x104HEK293cellsperwellinapproximately300-400µlofDMEM(10%FBS)inaPoly-L-Lysine48-wellplate.
•Afterovernightincubationat37˚C,wellsarecheckedtohaveapproximately70-80%confluency.
2.Changethemediaofeachwellwith200µlof10%FBSDMEMandplacebackintheincubatorfortenminutestocomebackuptotemperature.
3.Tomeasureinfectivity,werecommendperformingserialdilutionsofyourvirussupernatantacrossmultiplewells.Inthefirst3wellsadd50µlofvirussupernatant,inthesecondrowaddeithera5-or10-folddilutionandrepeatthisacrossmultiplerowsintheplate.Theaimistoachieveapproximately10-20%infectedcells,significantlymoreorlesslevelsofinfectivitycanresultininaccuratetitrecalculations.
4.Ensuretoleavesomewellsuninfectedtoactasnegativecontrols.
Incubatetheplatefor72hours.
5.Ifthelentivirusvectorcontainsareportergenetheninfectivitycanbemeasuredbyflow-cytometry.
6.Titrecanbeworkedoutbydetermining:
7.VolumeofsupernatanttestedinmL
%ofcells/100xnumberofcellsinitiallyinthewell
IPStatus:
ThisproductcontainsOxfordGeneticsproprietarysequencestoimprovetheexpressionofeachviruscomponent.Thereagentcanbeusedtopackagelentivirusgenomesforcommercialapplicationssubjectagreeingtoourlimitedusagepolicy.
Thismeansthatvirusparticlescanbegeneratedforcommercial(non-bioproduction)applications,however,thereagentcannotbesequenced,transformedintobacteria,orreverseengineered.Anysuchactionwouldcontravenethelimitedusagepolicyofthisproduct.
Forapplicationsinvolvingbioproductionmanufactureoflentivirusparticlespleasecontactusforfurtherdetails.
