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.
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(forced expression)或过表达(overexpression),以观察基因表型,反义则是用来抑制基因表达,以观察在目的基因的表达受到抑制的情况下表型的变化
我现在手上有PMD2.G与PSPAX2两个载体，求推荐与其配套的慢病毒过表达载体，谢谢。
常用细菌质粒进行构建，构建过程中运用限制性核酸内切酶切割出与目的基因相合的末端（多为黏性末端，也有平末端），采用DNA连接酶连接，导入生物体实现表达。标记基因可帮助识别质粒并检测是否成功整合到染色体DNA中。
表达载体（Expression vectors）就是在克隆载体基本骨架的基础上增加表达元件（如启动子、RBS、终止子等），使目的基因能够表达的载体。如表达载体pKK223-3是一个具有典型表达结构的大肠杆菌表达载体。其基本骨架为来自pBR322和pUC的质粒复制起点和氨苄青霉素抗性基因。在表达元件中，有一个杂合tac强启动子和终止子，在启动子下游有RBS位点（如果利用这个位点，要求与ATG之间间隔5-13bp），其后的多克隆位点可装载要表达的目标基因。
常用细菌质粒进行构建，构建过程中运用限制性核酸内切酶切割出与目的基因相合的末端(多为黏性末端，也有平末端)，采用DNA连接酶连接，导入生物体实现表达。标记基因可帮助识别质粒并检测是否成功整合到染色体DNA中。
表达载体(Expression vectors)就是在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。如表达载体pKK223-3是一个具有典型表达结构的大肠杆菌表达载体。其基本骨架为来自pBR322和pUC的质粒复制起点和氨苄青霉素抗性基因。在表达元件中，有一个杂合tac强启动子和终止子，在启动子下游有RBS位点(如果利用这个位点，要求与ATG之间间隔5-13bp)，其后的多克隆位点可装载要表达的目标基因。
各位好，最近构建慢病毒沉默载体，问题是提完质粒酶切验证是阳性，但是测序检测不到插进去的shRNA，已经重复连接转化多次，依旧没有得到阳性质粒，请大神帮忙分析下原因，感谢！！
问题
1AmpR是在筛选阳性质粒时发挥作用；
2氨卞青霉素只对原核生物起作用；
3在病毒颗粒感染目的细胞后，AmpR并未在目的细胞中表达（或者说没整合入目的细胞基因组）
4如果有整合入目的细胞基因组，那么可以起到药物筛选的作用吗？
慢病毒系统构建.doc(182.0k)
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