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. 

Oxford Genetics公司位于英国牛津，由来自生物各领域中具有多年基因工程经验的专家组建而成，专注于为全球生物不同领域的科研用户和生物技术工业用户提供多种DNA表达载体。 其所有产品基于一个中心质粒骨架(SnapFast )SnapFast 载体经过精心设计，不同的构建中配以不同的DNA元件，在特异酶切位点更换需要的元件，可进行上千种改造，满足各种生物学研究的需要。该公司还为科学家提 供万能克隆平台和DNA插入匹配库，有针对哺乳动物，细菌和噬菌体等多个系统的各种元件，包括启动子、报告基因、抗性标记、标签蛋白、核糖体位点、复制起 点、多克隆位点、融合蛋白、信号肽、终止子及其他DNA元件(SV40原点、P2A肽段、CMV增强子、小启动子等)。所有的元件都可以在不同载体间互换 使用，并且和很多市场上应用的克隆载体和穿梭载体都兼容，利于基因片段的转移。该系统非常灵活、高效、经济、知识产权友好，可解决目前基因工程研究中普遍 存在的克隆问题。