Virus Protocol - Generating Stable Cell Lines - Addgene

This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. Skiptomaincontent Protocols GeneratingStableCellLineswithLentivirus GeneratingStableCellLineswithLentivirus Youmayalsolike... ViralVectorGuides VirusBlogPosts MolBioProtocols ViralService Introduction Thisprotocolcanbeusedtogeneratestablecelllinesexpressingageneofinterestfromanintegratedlentiviralvector.Unliketheshorttermproteinexpressionobservedusingtransienttransfectionapproaches,generatingcelllinesusinglentiviralvectorsenableslong-termproteinexpressionstudies.Moreover,repeatingexperimentsinastablecelllineasopposedtotransiently-transfectedcellsincreasesreproducibility,asiteliminatesthevariationassociatedwithrepeatedtransienttransfection. Somelentiviralvectorsdelivermammalianantibioticresistance(e.g.,puromycin,blasticidin),whichenablesselectionofastablecellcultureaftertransduction.Performingantibioticselectionontransducedcellsenableseliminationofuntransducedcells,resultinginamorehomogenous(butstillpolyclonal)cellpopulation.Dependingonthetransducibilityofthecelllineused,thisantibioticselectionmaybeavitalstepforobtainingapopulationofcellsthathavetakenupthelentiviraltransgene.Notethatnotalllentiviralvectorsdeliverantibioticresistance. ThisprotocolwasestablishedusingLenti-X293Tcellsbutcanbeadaptedtoalternativecelllines. LastUpdated:January19,2019 WorkflowTimeline Day0:SeedandTransduceCells Day2-3(am):Removemedia,replacewithfreshmediacontainingselectionreagent Day3-14:Changemediaasneeded Day14-18:Expandandharveststablecelllines Equipment Biosafetycabinet Pipetman Pipettors Incubator Reagents DMEMhighglucose L-alanyl-L-glutamine(oralternativestableglutamine) Heat-inactivatedFBS Polybrene PBSpH7.4withoutcalciumormagnesium(cationscanaffecttheattachmentofadherentcells) Microcentrifugetubes 6-welldishes Pipettes Pipettetips Titeredlentiviruscontainingyoursequenceofinterest ReagentPreparation DMEMComplete:10%v/vFBSand4mML-alanyl-L-glutamine Toa500mLbottleofDMEMhighglucose,add55mLofheatinactivatedFBSand11mLof200mML-alanyl-L-glutamine.Storeat4°C. *Pro-Tip*DifferentbrandsandFBSlotscanpromoteorinhibittransfection.TestavarietyofbrandsandFBSlotnumberstofindonesuitablewithyourprotocols.FBScanbepurchasedalreadyheatinactivatedoritcanbeinactivatedinthelabbyheatingto56°Cfor30minutes. ConsiderationsBeforeYouStart Thehealthofthethetargetcelllineiscriticalforobtainingaccurateresults. Checkthecellsformycoplasmaregularly Donotoverorunder-growyourcells. Thawanewvialofcellsafter20-30passages. Donotaddpenicillin/streptomycintothemedia. Infectionefficiencywillvarybetweencelllines. Itisnotrecommendedthatlentiviralsupernatantsbesubjectedtomultiplefreeze-thawcycles. Procedure Beforebeginning,determinetheoptimaldoseofselectivereagentforyourtargetcellline.Todothis,treattargetcellswitharangeofdosesofantibioticanddeterminethelowestdosethatkillsallofthecells. PrepareabatchofDMEMcomplete+10µg/mLpolybrenebydiluting20µLof10mg/mLpolybreneinto20mLmedia. Rapidlythawthelentiviralaliquotat37°C. PreparearangeofdilutionsofthelentivirusinDMEMcomplete+10µg/mLpolybrene.Note,thisisjustasampleofpossibledilutions.Youmaywanttotryhigher/lowerdilutionsdependingonyourdownstreamapplications.Ifyou’vetiteredyourvirusbeforehand,youcannarrowthisrangeaccordingtotheresultsofyourtitration. Mixthedilutionswell. Dilution VolumeofLentivirus(μL) VolumeofDMEMcomplete+10µg/mLpolybrene(µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add0.5mLofasingleviraldilutiontoeachwell(eachwellgetsonedilution,soa6-wellplatewillhold5dilutionsplusone'novirus'controlwell). Performa"reversetransduction"byseeding50,000cellsintoeachwellofthe6-welldish.Thesecellswillbeaddedtothewellsthatalreadycontain0.5mLofvirussolutionsatvariousdilution.Makesuretousethepolybrene-containingmediatomakethecellsolutioninthisstep.Toseedthecells: Prepareabatchofcellsasfollows:Dilute350,000cellsintoatotalvolumeof7mLofDMEMcomplete+10µg/mLpolybrene. Mixwellbypipettingorinvertingthetube. Aliquot1mLofcellsuspension(i.e.,50,000cells)intoeachwellofthe6-welldish.Thisbringsthetotalvolumeineachwellupto1.5mL.SinceallthemediainthesewellswasmadewithDMEMcomplete+10µg/mLpolybrene,thefinalconcentrationofpolybreneineachwellshouldbe10µg/mL. *Pro-Tip*Transducingtoomanycellsrelativetothenumberofvirusparticlesreducesthetransductionefficiency,resultinginmassivecelldeathuponantibioticselection.Thenumberofcellstransducedshouldbeenoughthattheycangrowoutinareasonableamountoftime,butnotsomanythattheyvastlyoutnumberthevirusparticles. Incubatethecellswiththevirusfor48-72hours. Gentlyaspiratethemediafromthecells. Add1.5mLofDMEMcompletecontainingtheappropriateantibiotic.Thisisthebeginningoftheselectionprocess,whichwillbegintheselectionofastablecellpool. Observethedisheverydaytoensurethatthecellsintheuntransducedwell(0µLlentivirus,above)aredying.Performregularfluidchangesthemonitorthegrowthofthecells. *Pro-Tip*Dependingontheefficiencyofyourtransduction,youwillseedifferentdegreesofcelldeathuponantibioticselection.Itisimportanttomonitorthesecellsregularlyandreplacethecellmedia.Celldeathbysomecellsintheculturemayadverselyaffectthesurvivingcellsintheculture,soitisimportanttodoregularfluidchangesandmaintainoptimalgrowthconditionsforthesurvivingcells.Evenintheabsenceofcelldeath,thecellmediashouldbechangedevery2-3daystomaintainthedoseofantibiotic,whichmaynotbestableat37°C. *Pro-Tip*Toachieveastablecellpool,theantibioticselectionshouldlastatleastaslongasittakesthecontrol(untransduced)cellstocompletelydie.Afterthat,thecellsmayundergoadditionalselecitonwhilethepopulationexpands.Atthistime,someresearchersreducetheconcentrationoftheantibioticinculture,orremovetheantibioticentirely.Iftheantibioticisreducedorremovedfromtheculture,checkthecellsregularlytoconfirmtransgeneexpression. Aspolyclonalpopulationsofresistantcellsstartcomingthroughandtheindividualwellsbecomeconfluent,expandintolargervessels.Aconfluentwellofa6-welldishcanbeexpandedintoa10cmdish.Aconfluent10cmdishcanbeexpandedintotwo75cm2flasks. *Pro-Tip*Thisselectionmethodresultsinapolyclonalcellpopulation,meaningthatthetransgenehasintegratedindifferentlocationsinthevariouscellsintheculture.Thisisbecauselentiviralintegrationisrandom.GiventheMOIofthelentivirusused,cellsmayalsoexhibitvaryingnumbersofintegrationevents.Inotherwords,ifanMOI>1wasused,somecellsmayhave1copyofthetransgene,whileothershave>1copyofthetransgeneintheirgenome.Thiscanresultinvaryingexpressionlevelsofthetransgenefromdifferentcellsinthepopulation. Oncethepolyclonalpopulationsaregrowingwellandhavebeensufficientlyexpanded,preparecellstocksand/orharvesttotestforproteinexpression. Typicallycellstransducedwithlowerdilutionsoftheviruswillhavehigherlevelsofexpression.Considerexpandingpopulationstransducedwithavarietyofdilutionsandpickthepopulationthathasthemostdesirablelevelofexpression. Overtime,transgeneexpressioninapolyclonalpopulationmaydrop.Thisisbecausecellsthatexpresshighlevelsofthetransgenemayhavereducedgrowthrates,especiallyifthattransgeneistoxic.Eventually,therapidlygrowinglow-leveltransgeneexpressorsmaytakeovertheculture.Toovercomethis,considergeneratingmonoclonallinesfromtheearlypolyclonalpopulations. SampleData Figure1:GenerationofmonoclonalcelllinesfromexpansionofindividualA549cellsstablyexpressingCas9.A549cellsweretransducedwithlentiCas9-Blastandthenselectedwith1µg/mLblasticidinfor9days.Singlecellswerethenplatedinindividualwellsofa96-wellplateandleftundisturbedfor13days.(a,b,c)Coloniesformedbyexpansionofsinglecellsfor13days.lentiCas9-BlastwasagiftfromFengZhang(Addgeneplasmid#52962)andisdescribedinImprovedvectorsandgenome-widelibrariesforCRISPRscreening.SanjanaNE,ShalemO,ZhangF.NatureMethods.2014Aug;11(8):783-4.