Selection and Expansion of Clones Post-Transfection - Mirus Bio

Step 3:Stable Cell Line Generation-Selection/Expansion of Clones; includes ID single clones, transfer, assess expression, freeze down high expressing.     Applications:StableCellLineGenerationStableCellLineGenerationSTEP1:AntibioticKillCurveSTEP2:StableTransfectionSTEP3:Selection/ExpansionProducts SelectandExpandClonesPost-Transfection At48-72hoursposttransfection,addtheselectionantibioticatthehigh,optimalandlowdosetoeachofthetransfectedT75flasks Asacontroltoassesstheantibioticresponsesidebysideinnon-transfectedcellsinthe6-welltissuecultureplate,addtheantibioticatthesameconcentrationsthatareusedfortheT75flasks(asindicatedinthetablebelow).Includeanoantibioticcontrol(seetablebelow) Changemediacontainingselectionantibioticevery2-3days.Examinethecellsforvisualtoxicitydaily.Typically,mostofthecellsthathavenotintegratedthetransfectedplasmidwilldiewhilethecellsthathaveundergoneplasmidintegrationwillsurviveby9dayspost-transfection.Sincetheefficiencyofstableintegrationintothegenomeisquitelow,survivingcellsshouldbeallowedtoexpandintheT75flasktoensurethattheselectedclonesarenotunstable KeepreplacingmediacontainingselectionantibiotictwiceaweekuntilthecellsintheT75flaskreachhighconfluence.Atthispoint,theycanbefrozendownasapolyclonalline WellNo. Antibioticdose 1 Noantibiotic 2 Lowdoseofantibiotic 3,4 Optimaldoseofantibioticineachwell 5,6 Highdoseofantibioticineachwell PolyclonaltoMonoclonalSelection Thepolyclonalcellculturecanbefurtherprocessedtoisolatemonoclonesusingdifferenttechniquessuchas: Limitingdilution:Thegoalusingthismethodistoisolateeachindividualcellthatcarriesselectionbyplatingthematverylowcelldensities(<1wellperwellin96wellplates)andexpandcoloniesfromthosesinglecellsinseparatewells.Thisisacost-effectiveyettediousprocess;additionally,certaincelltypesdonotsurvivethelimiteddilutionstepduetoaneedforsecretedfactorsfromneighboringcells.Analternativeinsuchcasesistouseconditionedmediaand/or2Xserumtoincreasecellattachmentandsurvival.Occassionally,culturingcellsinsemi-solidmediasuchassoft-agarandmethylcellulosemighthelp,particularlyincaseofsuspensioncells Cloningringsandtrypsindiscs:Iflimiteddilutiondoesnotwork,thecloningprocessmightneedtobecarriedoutathighercellconcentrationsandrepeatedafewtimestoensuremonoclonality.Usingcloningringsand/ortrypsindiscsisaviableoptionforadherentcellsinthisscenario.Inthismethod,selectedcellsareseededsparselybutnotatlimitingdilutionin10cmdishesandallowedtoexpandandformdiscerniblecoloniesfor2-3weeks.Theindividualcoloniescanthenbetrypsinizedandtransferredtoanothersmallerculturevesselusingeithercloningringsortrypsindiscsformonoclonalexpansion Fluorescenceactivatedcellsorting(FACS):Thismethodcanbeemployedifadetectablemarkerisexpressedonthecellspost-transfection.SinglecellscanbeisolatedusingFACsandreplatedtogenerateamonoclonallineage Automatedclonepicking:Moresophisticatedinstrumentationbasedmethods,e.g.ClonePix™technology(MolecularDevices)allowsacompletelyautomatedprocessofhighproducercloneidentificationandexpansion Ofalltheabove-mentionedmethods,limitingdilutionisthemostcost-effectiveandfrequentlyadoptedtechnique;adetailedprotocolusinglimitingdilutiontogeneratemonoclonalcelllinesisasfollows: Identifysingleclonesbylimitingdilutionandexpansion Platethepolyclonalcellsfromtheselectionstepatadensityof10cells/mlina96-welltissuecultureplateadding100µlperwell(i.e.,1cellperwell) Assessthenumberofcellsperwellafter18-24hoursandnotethewellswithonly1cell Afterthe4thday,assessthenumberofcoloniesperwellinthewellsthatonlyhadonecellattheinitialassessment.Assumeeachcolonyisclonal.Onlywellswith1colonyperwellshouldbeconsideredmonoclonal Afterthesewellsareidentified,continuetoverifycolonynumbereveryweekuntilthewellhasreachedhighconfluence.Note:Ifamonoclonalpopulationishighlycriticalforyourexperimentalset-up,thedilutionstepcanberepeatedasecondtime.Repeatingthelimiteddilutionstep3-4timesensuresthattherearenofalse-positivemonoclones 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.Acommonfreezingmediumis10%DimethylSulfoxide(DMSO)plusnormalgrowthmedium Uponestablishingyourtargetmonoclonalstablecellline,aloweramountofantibioticcanbeusedformaintenance.Itiscriticaltofollowthepassagenumberofstablecelllinessincethestabilityofclonalcelllinesmightvary.Someclonesmayloseexpressionafterseveralpassages.Freezedownsamplesfromearlypassagetoprolongtheiruseafterthawing STEP1:AntibioticKillCurve STEP2:StableTransfection Forstablecelllinegeneration,useMirus'highqualitycell-culturegradeselectionantibioticsthatareeasy-to-use: AllMirus'broadspectrumplasmidDNAtransfectionreagentscanbeusedforstablecelllinegeneration.Visittheproductpagesformoreinformationoneachproduct: TransIT-X2®DynamicDeliverySystem TransIT®-2020TransfectionReagent TransIT®-LT1TransfectionReagent Don'tSeeYourCellType?ConsultReagentAgent®TransfectionDatabase 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