Protocol of Stable Cell Line Generation - Creative BioMart

The generation of stably-transfected cell lines is critical for a wide range of applications. It is applied to the production of recombinant proteins, gene ... ProtocolofStableCellLineGeneration September27-30,2022 BostonConventionandExhibitionCenter Booth#336 LearnMore 00DAYS 00HRS 00MINS 00SECS September27-30,2022 BostonConventionandExhibitionCenter Booth#336 LearnMore 00DAYS 00HRS 00MINS 00SECS October17-20,2022 BOSTON,MA&VIRTUAL Booth#615 LearnMore 00DAYS 00HRS 00MINS 00SECS October16-19,2022 BostonConvention&ExhibitionCenter Boothnumbertobeupdated LearnMore 00DAYS 00HRS 00MINS 00SECS Nov.8–Nov.12,2022,Boston,MA 37thAnnualMeeting&Pre-ConferencePrograms Boothnumbertobeupdated LearnMore 00DAYS 00HRS 00MINS 00SECS HOME PRODUCTS RecombinantProteins PROTACTargets FullLengthProtein MonkeypoxVirus-relatedProteins Fluorescent-labeledRecombinantProteins Avi-tagBiotinylatedProteins Car-TCellTargets LabeledCar-TProteins ProteinKinases Biomarker Cytokines ImmuneCheckpointProteins CDAntigens SARS-CoV-2ProteinsandTheirTargetProteins Heavy-LabeledProteins NativeProteins Cell&TissueLysates ChromatographyReagents GMPProteins Lectins AssayKits Others SARS-CoV-2 SERVICES CustomProteinService ProteinAnalysis Screening&Profiling BiopharmaceuticalSolution Food&HealthTest RESOURCE ResearchArea SuperFamily Principle&Protocol SignalPathway ProteinConversions DownloadCenter ORDER PriceInquiry OnlineOrder FAQs COMPANY AboutUs ContactUs Distributors Promotion Events Career KeywordsSearch GeneSearch GoogleSearch RESOURCE ProtocolofStableCellLineGeneration Home/Resource/ Principle&Protocol/ ProtocolofStableCellLineGeneration ProtocolofStableCellLineGeneration Background Thegenerationofstably-transfectedcelllinesiscriticalforawiderangeofapplications.Itisappliedtotheproductionofrecombinantproteins,genefunctionstudies,aswellasdrugdiscoveryassays.Stableexpressionoftargetgeneincelllinesovercomesthelowtransfectionefficiencyoftransientexpressionandproducesmoretargetproteins. Therearetwotypesofstablecelllines.Oneisachievedbyeukaryoticvectorsthatharborelementsforepisomalmaintenanceinthenucleusofatransfectedcell.Anotherisachievedviadirectintegrationofthetransfectedplasmidintothetargetcellsgenome.Episomalstabilityisoftenlimitedandepisomalplasmidelementsisoftenrestrictedtocertainspecies,thereforeintegrationintothehostcellchromosomeisincommonuse.Althoughintegrationintothehostcellchromosomeisarareeventand,formostpurposes,clonaleventshavetobeisolated,stabilityoftheintendedgeneticmodificationusuallyismuchhigher. Majorchallengesforgenerationofstablecelllinesarelowtransfectionefficiencyand/orintegrationfrequency.Stableexpressioncanbeinfluencedbythetransfectionmethodused.Thetransfectionmethoddeterminesthecelltypeforstableintegration.ItisknownthatliposomereagentscanbeusedtotransferDNAintoadherentcelllines.WhileviralmethodsorelectrotransfectionareusedtodeliverDNAintoprimarycellsornotoriouslydifficult-to-transfectsuspensioncelllines.Unfortunately,viralmethodssufferfromseverallimitations,suchastimeconsumingproductionofvectorsandsafetyconcerns,whileelectrotransfectionsufferfromthelowcellsurvivalrate. SelectionMarker Inordertoselectstably-transfectedcells,aselectionmarkermustbeco-expressedwiththetargetprotein.Themarkergenecouldbeoneitherthesameplasmidvectororasecond,co-transfectedvector.Thereareavarietyofsystemsforselectingtransfectedcells,includingresistancetoantibioticspuromycin,neomycin,DHFR,andglutaminesynthetase.Aftergenetransfer,cellsaredevelopedinmediumcontainingtheselectiveagent.Onlythosecellswhichhavecontainedthedrugresistantgenesurvive. MethodstoGenerateStableCellLines Dependingonthescopeoftheexperiment,severaloptionsareusedforthegenerationofastablecellline.Amixedpopulationofdrugresistantcellscanbeuseddirectlyforexperimentalanalysiswiththeadvantageofgeneratingfastresults,butalsothedisadvantageofdealingwithanundefinedandgeneticallymixedcellpopulation.Anotheroptionistogenerateamonoclonalcellline.Inthismethod,itisnecessarytodilutetheresistantcellsbyplatingin96-wellplatesinsuchawaythatcultureassingleandisolatedcells.Subsequently,thecloningofsinglecellmayberepeatedseveraltimestoobtain100%clonalpurity.Thisculturemethodcanbeusedforscreeningexperimentsorconductionstudiesbyusingahomogenousanddefinedcellsystem. Inconclusion,dependingonthetypeofexpressionyou’reinterestedinandtheconstructthatyouareincorporating,therearemanydifferentapproachesforgenerationofstablecelllines.ThisprotocolisspecificforthegenerationofamonoclonalcelllinethatresistancetoantibioticsG418(neomycin).Theendresultthatyouarelookingforisapopulationofcellsinwhich100%ofcellsareexpressingyourfusionprotein. CultureConditions Cultureconditions(passage,splitrhythm,number,etc.)ofyourselectedcelltypearecriticalforgenerationofstablecelllines.Foroptimalresults,werecommendfollowingthecellculturerecommendationsofthesupplier(e.g.ATCC)fortherespectivecelltype.Ingeneral,forpromotinggoodproliferationandcellphysiology,thecelllineshouldbepassagedtwodaysbeforetheexperiment.Besides,cellpassageshouldnotbehigherthan30duetothepossibilityofinterferencewithintegrationefficiency. ExperimentalOutline Designexperiment Designexperimentandchooseexpressionvector,celltypeandtransfectionmethod.Makesurethatyourexpressionvectorandtransfectionmethodaresuitableforyourcelltype. ChoosetheG418concentrationandcellnumber DetermineappropriateG418concentrationandcellnumberperwellbymatrixtitration.PayattentiontotheactiveconcentrationofstockG418isvaryfrombatchtobatch. Transfection Transfectexpressionplasmidintocells.Peasefollowthemanufacturer’sinstructionofyourtransfectionsystem.DonotaddG418toculturemediumimmediatelyaftertransfection. Cellselection PlatetransfectedcellsandcultivatecellsinmediumwithG418inappropriateconcentration.Amixedpopulationofdrugresistantcellsisobtained. Monoclonalcelllinescreening Dilutecellsinto96wellcultureplatesinappropriatecelldensityperwell.Feedevery10dayswithselectionmedium.Refreshedselectionmediumisimportanttoavoidfalsepositivecells. Analyze Makesuretochooseasuitablemethodforyourapplicationtoanalysisyourstable-transfectedcells.   Protocol 1.ChoosetheG418Concentration SusceptibilitytoG418isdifferentamongcelllines,whichmanyevenvarywithcellpassagenumbers.Theselectioncondition(e.g.G418concentration,platingdensity)foryourspecificcelltypeneedstobedeterminedexperimentally.DeterminetheminimumlevelG418concentrationtoguaranteetheminimumimpacttocellgrowth.NotethattheactiveconcentrationofstockG418canvaryconsiderablyfrombatchtobatch.Therefore,werecommendtestingG418sensitivityforeverynewbatch.ThefinalplatingdensitydependsonthespecificcelltypeandtheG418concentration.WethereforerecommendmatrixtitrationofG418andtitrationofcellnumberfordeterminationofplatingdensityinone96-wellplate. Pre-plate100μlmediumineachwelloftheplate. Add100μlofcellsuspensioncontaining4000cellsperwelltothefirstcolumn(#1). Aftergentleupanddownpipetting,carryover100μltothenextcolumn,therebydilutinginaratioof1:2.Repeatthisprocedureforeachconsecutivecolumn. Discard100μlfromthelastcolumn(#12)aftercompleting.Thefirstcolumnshouldthencontainabout2000cellsperwell,thelastcolumncontainaroundonecellperwellonaverage. Add100μlofG418containingmedium(2.8mg/ml)tothefirstrow(A)forafinalG418concentrationof1.4mg/ml. AddG418tothefollowingrowsindecreasingconcentrationsofG418instepsto0.2mg/ml.Forthelastrow(H)addmediumwithoutG418. Incubatecellsatstandardconditions. Analyzecellgrowthbymicroscope.Insomecases,cellgrowthcanalsobeobservedbychangeofmediumcolor. Ifyouobservecellgrowth(after10days)inthewellswithoutG418,itisreasonabletoassumethatthosecellscangrowoutstartingassinglecells. ChoosetheG418concentrationwhichisjustabovetheonewhichshowscompletecelldeathastheappropriateG418concentrationforselection. 2.Transfection Fortransfection,pleasefollowthemanufacturer’sinstructionofyourtransfectionsystem.Theimportantthingistotransfecttheexpressionplasmidcontainingthetargetgeneandthesequenceforadrugresistancegeneintoyourcells.Wesuggestsettinganegativecontrolofuntransfectedcellsforselection.Besides,itismuchbettertocheckthetransfectionefficiencyandintegrationfrequencyofyourexperimentwithaGFP-controlplasmid. 3.CellSelectionPost-Transfection Aftertransfection,allowcellstogrowandtoexpresstheproteinforG418resistanceundernon-selectiveconditionsforabout24-48hours(reachthepeakofproteinexpressionaccordingtoyourtransfectionsystem). Usesuspensioncellsdirectlyortrypsinizeadherentcellsforanalysis.Analyzefortransfectionefficiency24-48hourspost-transfectionbymicroscopyorwesternblotofyourtargetproteinandpositivecontrol. Countlivingcellsviatrypanbluestainingorotherappropriatemethods. UsingstandardmediumandtheappropriateamountofG418pretestedforyourcelltype,platecellsina96-wellplatewithdifferentcellnumbersperwell(e.g.,0.5,1,2,5,10)inavolumeofatleast100μlperwell.Dependingoncellconcentrationdeterminedbefore,conductseveralserialdilutionstepsasapplicable.Itisimportanttothoroughlysuspendcellsbeforeseeding,butavoidharshtreatmentbyfrequentpipetting. Incubatecellsunderselectiveconditionsandfeedcellsregularlywithfreshselectionmedium. Cellclonescanbeanalyzedorfurtherexpandedassoonascellsinthenon-transfectedcontrolwellshavecompletelydied. Inordertohelpassuringthatselectedcellpopulationsareclonesderivedfromasinglecell,anotherroundoflimitingdilutionunderselectionisrecommended. 4.AnalysisofStableCellLines Onceyouhaveobtainedresistantcelllines,youshouldexpandthecellsandassayyourtargetgenecomparedwithpositiveandnegativecontrol.Youcandetecttheexpressionoffusionproteinbyanappropriateanalysismethodsuchaswesternblot,microscopy,ELISA,aswellasflowcytometry. CreativeBioMartprovidesstablecelllineservicestomeetyourspecificneeds.Notonlyhavewedevelopedcelllinesstableatexpressionlevel,butalsoestablishedstablecelllinesreadyforassaydevelopment. Tags:None ApplyForACoupon $50OFFYourFirstPurchase ApplyForaCoupon Enteryouremailheretosubscribe. submit creativebiomartinc. GlobalLocations Tel:/ Fax: Email: Easyaccesstoproductsandservicesyouneedfromourlibraryviapowerfulsearchingtools. FollowUs Copyright©2022CreativeBioMart.AllRightsReserved.TermsandConditions|PrivacyPolicy