Generate Stable Cell Lines With Chemical Transfection

Stable cell line generation is made possible by the use of positive selection markers such as hygromycin, G418/Geneticin, zeocin, and blasticidin antibiotic ...   Ourproductsspeakforthemselvesinqualityandperformance.REQUESTAFREESAMPLE TipsfromtheBench:TransfectionTip GenerationofStableCellLinesMostcellbiologyexperimentsutilizetransienttransfectionprotocolsthataffordpeakgeneexpressionbetween24-96hoursposttransfection.However,ifsustainedgeneexpressionisrequiredforlongerperiodsoftime,generationofstablecelllinesisaviableoption.Furthermore,stablecelllinesselectedthroughlimiteddilutionprovideageneticallyhomogenousandclonalpopulation. Stablecelllinegenerationismadepossiblebytheuseofpositiveselectionmarkerssuchashygromycin,G418/Geneticin,zeocin,andblasticidinantibioticresistance.Selectionmarkerscanbedeliveredusingthesameplasmidthatcontainsthegeneofinterest(incis),oronaseparateplasmid(intrans)thatneedstobeco-transfectedwiththeplasmidcontainingthegeneofinterest.Thecisapproachisgenerallyeasierandhasahigherlikelihoodofproducingdrug-resistantstabletransfectantsthatexpressthegeneofinterest.Thetransmethodofco-transfectionisagoodalternativeininstanceswherethetargetconstructdoesnothavetheantibiotic-resistancegeneinthevectorbackbone.Insuchcases,aplasmidmixturecontaining5to10partsgeneexpressionplasmidand1partantibioticselectionmarkerplasmidcanbeintroducedintocells.Thisplasmidratiohelpsensurethattheselectedcellswillexpressboththegeneofinterestandtheselectionmarker. Stablecelllinegenerationprotocol Theprotocolforgeneratingstablecelllinesrequiresseveralstepsasshownbelow: Generateakillcurvetodeterminetheoptimalselectionantibioticconcentration Transfectcellswithdesiredplasmidconstruct(s) Selectandexpandstablepolyclonalcolonies Identifysingleclonesbylimiteddilutionandexpansion Transferclonesandassessexpression Expandandfreezedownhighexpressingclones Generateakillcurvetodeterminetheoptimalselectionantibioticconcentration Thefirstcriticalstepforstablecelllinegenerationisdeterminingtheoptimalantibioticconcentrationforselectingstablecellcolonies.Akillcurveisadose-responseexperimentwherethecellsaresubjectedtoincreasingamountsofantibiotictodeterminetheminimumantibioticconcentrationthatisneededtokillallthecellsoverthecourseofoneweek.Performingakillcurveisrecommendedwitheachnewcelltypeorwhenanewselectionantibioticordifferentlotofselectionantibioticisused. Platecellsin0.5mlcompletegrowthmediumperwellina24-welltissuecultureplateonedaypriortointroducingantibioticselection.Ideallycellsshouldhavereachedhighconfluence(~60-80%)priortoaddingtheselectionantibiotic.Typicalcelldensityrangesareasfollows: Adherentcells:0.4–1.2×105cells/well. Suspensioncells:1.6–2×105cells/well. AddincreasingamountsoftheappropriateantibioticsuchasG418toduplicatewellsofcellsplatedincompletemedia.Includeano-antibioticcontrol.Forexample,add0,50,100,200,300,400,500,600,700,800,900,and1000µg/mlselectionantibiotictoduplicatewellsofcellsplatedincompletegrowthmedia. Replacemediawithselectionantibioticevery2-3daysforuptoaweek.Examinethecultureeverydayforsignsofvisualtoxicity.Determinethefollowingantibioticdoses: Lowdose-theantibioticconcentrationatwhichminimalvisualtoxicityisapparentevenafter7daysofantibioticselection Optimaldose-thelowestantibioticconcentrationatwhichallcellsaredeadafteroneweekofantibioticselection Highdose-theantibioticconcentrationatwhichvisualtoxicityisevidentwithinthefirst2-3daysofantibioticselection(allcellsdeadby7days) Transfectcellswithdesiredplasmidconstruct(s) Whileperformingthekillcurve(1week),optimizetransfectionconditionsinaT75flaskbytransfectingareporterplasmid(suchasaGFPencodingplasmid)intocellsathighconfluence.Determinetheappropriatedoseofplasmid(5-15µg)andtransfectionreagent(15-45µl)inaT75flask.Observereportergeneexpression(GFP)andtoxicityatregulartimepointsoveratleasta48hourperiod,optimallyfor72hours.SpecifictipsonoptimizingDNAtransfectioncanbefoundhere.UsetheoptimalDNAandtransfectionreagentdosageforgeneratingstabletransfectants. Optional:Linearizeyourtargetplasmidsbeforetransfection.Whengeneratingastablecellline,thetransfectedplasmidundergoesrecombinationduringchromosomalintegration.Therecombinationeventcanoccurwithinanyregionoftheplasmid,includingthegeneexpressionorselectablemarkercassettesthatmightdisrupttheirfunction.Toincreasethelikelihoodthatrecombinationwilloccurinnon-essentialplasmidregions,suchasthebacterialrepliconorbacterialmarkergene,linearizetheplasmidwithrestrictionenzyme(s)thatcutwithinthesenon-essentialregions.Priortotransfection,purifythelinearizedDNAbyethanolprecipitationorcolumnpurification. Foreachindividualstablecelllinetobecreated,platecellsinthreeT75flasksandone6-welltissuecultureplateapproximately18–24hoursbeforetransfectionsuchthattheyreachhighconfluence(~60-80%)atthetimeoftransfection.Typicalcelldensityrangesareasfollows: Adherentcells:0.8–2.4×105cells/mlofcompletemedia. Suspensioncells:3.2–4×105cells/mlofcompletemedia. Leavethe6-welltissuecultureplateuntransfected.Thiswillserveasanuntransfectedcontrol. Transfecttheplatedcellswith5-15µgoftotalplasmidDNAperT75flask.Iftheantibioticselectionmarkerisonaseparateplasmidthanthegeneofinterest,thenmaintaina10:1ratioof“geneofinterest”plasmidover“antibioticselection”plasmid.  Donotexposecellstotheselectionantibioticuntil48-72hoursposttransfectiontoavoidlowcellviability.Amediachangecanbeperformedat24hoursposttransfection,ifneeded. Selectandexpandstablepolyclonalcolonies At48-72hoursposttransfection,addtheselectionantibioticatthehigh,optimalandlowdosetoeachofthetransfectedT75flasks. Asacontroltoassesstheantibioticresponsesidebysideinuntransfectedcellsinthe6-welltissuecultureplate,addtheantibioticatthesameconcentrationsthatareusedfortheT75flasks(asindicatedinthetablebelow).Includeanoantibioticcontrol. WellNo. Antibioticdose 1 Noantibiotic 2 Lowdoseofantibiotic 3,4 Optimaldoseofantibioticineachwell 5,6 Highdoseofantibioticineachwell Changemediaevery2-3days.Examinethecellsforvisualtoxicitydaily.Typically,mostofthecellsthathavenotintegratedthetransfectedplasmidwilldiewhilethecellsthathaveundergoneplasmidintegrationwillsurviveby9dayspost-transfection.SurvivingcellsshouldbeallowedtoexpandintheT75flask. Replacemediawithantibiotictwiceaweek.WhenthecellsintheT75flaskreachhighconfluence,theycanbefrozendownasapolyclonalline.Cellscanalsobeplatedforselectionofsinglecellclonesusingtheprotocolbelow. Identifysingleclonesbylimiteddilutionandexpansion Platethepolyclonalcellsfromtheselectionstepatadensityof10cells/mlina96-welltissuecultureplateadding100µlperwell(i.e.,1cellperwell). Assessthenumberofcellsperwellafter18-24hoursandnotethewellswithonly1cell. Afterthe4thday,assessthenumberofcoloniesperwellinthewellsthatonlyhadonecellattheinitialassessment.Assumeeachcolonyisclonal.Onlywellswith1colonyperwellshouldbeconsideredmonoclonal. Afterthesewellsareidentified,continuetoverifycolonynumbereveryweekuntilthewellhasreachedhighconfluence.Note:Ifamonoclonalpopulationishighlycriticalforyourexperimentalset-up,thedilutionstepcanberepeatedasecondtime. Transferclonesandassessexpression Expandselectedsingle-colonywellsinthe96-welltissuecultureplatetohighconfluenceandtransfertoa12-welltissuecultureplate.GFPpositiveclonescouldbeassessedinthe96-welltissuecultureplate,butothersshouldnotbeassessedforexpressionyet(dependingonthereporterassay). Oncethe12-welltissuecultureplatecloneshaveexpandedtohighconfluence,theycanbepassagedtoa6-welltissuecultureplate.Asmallportionofthecellsshouldbeassessedforexpressionofthetargetproteinatthistimepoint. Propagateasmallportionofselectedcellsfor50-90doublingstoconfirmstabilityofexpressionbyverifyingexpressionofthetargetgeneatmultipletimepoints. Expandandfreezedownhighexpressingclones Onceexpressionisverified,clonesofinterestcanbescaleduptolargervolumes(e.g.aT75flask).Dependingonthecelltype,mostsinglecellclonesshouldreachhighcelldensitiesby2weeks(e.g.inHEK293cells);someslowgrowingclonescantakeupto4weeksforcompleteexpansion. Onceexpanded,freezedowncellstocksusingappropriatefreezingmediumlackingtheselectionantibiotic. Uponestablishingyourtargetmonoclonalstablecellline,aloweramountofantibioticcanbeusedformaintenance.Itiscriticaltofollowthepassagenumberofstablecelllinessincethestabilityofclonalcelllinesmightvary.Someclonesmayloseexpressionafterseveralpassages.Freezedownsamplesfromearlypassagetoprolongtheiruseafterthawing. TakemetoMirusBioproducts. GoBacktoTipsfromtheBench Close PleaseWait... Yourrequestisprocessing