The art of generating single cell clones - Bitesize Bio

A single cell clone is essentially generated from an original 'multiclonal' population, but has been separated from the rest in order to ... 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Makingmutationsinmammaliancelllinesisbecomingmucheasier,especiallywithadvancedmolecularengineeringtechniquessuchasCRISPR/Cas9,amongothers.However,aftermakingamutation,doyouknowifallofthecellscontainthesamemutationwiththesameexpressionprofiles,andarethereforehomogenous?Ifyouhave100%transfectionefficiencyusingaGFPreporter,forexample,thenatleastyouknowthatallofyourcellsweretargetedforthemutation.Andifallyoucareaboutisthatthemessageisknockeddown,thenawesternblotlookingatproteinexpressionmightalsosuffice.However,thecellsIgenerallyworkwitharedifficulttotransfect(muchlessthan100%transfectionefficiency),soIneedtoisolatesinglecellclonesfromWTclones.HowDoYouGenerateSingleCellClones?Asinglecellcloneisessentiallygeneratedfromanoriginal‘multiclonal’population,buthasbeenseparatedfromtherestinordertocreateapure,clonalpopulationthatisgeneticallyidentical.Thissoundsdifficult,butisactuallyquitepossible!Justalittletimeconsuming.BelowaretwomethodsthatIhavepersonallyusedtosuccessfullygeneratesinglecellclones.Serialdilutionmethod: Thismethodisexactlyasitsounds–youcreateaserialdilutioninordertogetdowntoapproximately1cell.However,itisprettytedious,andyoumustworkrelativelyquicklyatfirst.Afterthecellshaverecoveredfromatechniquetoinduceamutation,usetheserialdilutionmethodasfollows:Countthecellsinordertoput~16,000cellsintothefirstwell(A1)ofa96-wellplatecontaining200µLofmedium.Theother95wellsshouldcontain100µLoftherespectivemedium.(Performthisset-upwithatleast296-wellplatespermutationinordertogenerateatleast10-20clones).Remove100µLfromA1containingcellsandmixwithB1,pipettingupanddown3xtomixwellbeforeremoving100µLandmixingwithC1,etc.untilreachingH1,whichnowhas200µLofmedium.Quicklyadd100µLofmediumtoA1-G1(sothatallwellsincolumn1willcontainatotalof200µL).Usingamultichannelpipette,dilutethecells1:1acrosstherowsstartingwithcolumn1,mixupanddown3x,andthenmovetocolumn2andsoforth.Afterthedilutions,wellsarefilledwithanother100µLofmediumbeforebeingincubated.Allwellsnowhave200µLofmedium.Thisapproximatesthatatleast15wellsshouldcontain1cell.However,thisisnotalwaysperfectandthereforemonitoringthewellsareveryimportanttoensuring1cellperwell.TipsforMonitoringSerialDilutions:Ihavefoundthatsomeofthesinglecellsactuallydonotsurvive,andsometimescellsthatIdidn’tknowwerethereactuallyare.Therefore,Itendtomonitorwellsthatcontainanywherefrom0-3cells,whichareusuallyinthebottomrighthandcornerofthe96wellplate.Twodaysafterplating,Istarttoidentifywhichwellscontain0cells,1-3cells,and>5cells.Icontinuetofollowthewellscontaining0-3cells,eventuallydeterminingifanyofthesewellsreallycontainedasinglecell.Thiscanbearelativelyslowprocesswaitingfor1celltoproliferateenoughtofill1/3ofwellinordertotrypsinize.Inthemeantime,Iusuallyfeedthesewellswithspentmedium(mediumusedpreviouslybyhealthy,growingcellsandiscombined1:1withfreshmediumtoensureallthefactorshavenotbeenused)onceaweekandhaveobservedthatthishelpskeepthesinglecellclonesfromdying.Afterthesinglecellcloneshavebeenidentified,itusuallytakessometimebeforethecellscanproliferateintolargeenoughofaclonalpopulation(about1/3ofthe96well)tobetransferredtoalargersurfaceareatoreallygrowandexpand.Cloningrings/cylinders: Ihaveonlyusedthecloningringsafewtimesnow,butpreferthismethodovertheserialdilutionmethod.CloningringsarequitesmallandcanbemadefrompolystyreneorPyrex,andlookliketheendofaPasteurpipettewascutoff.BelowistheprotocolIuseforthecloningrings:Countthecellssothattherearenomorethan20cellsper10cmdish.Thisallowsthecellstobespreadoutinthedish.Thenextday,markthesinglecellsthathaveattachedtotheplateandseemfarenoughawayfromtheothercells.Remember:thecellswillneedtobemonitoredforclonalexpansion.Oncethereareafewsinglecellcoloniesinthedish,usethecloningringtoisolatethem.Todothis,carefullyaspiratethemediumfromthedish.Usingforceps,takethesterilizedcloningringthathasgreaseononesideandplaceitverycarefullyaroundthetinygrowingcolonyofsinglecellclones.Addapproximately40µLoftrypsinintotheringandincubatethecellsforaboutfiveminutesinordertocarefullydetachthecells.Washandresuspendthecellsintoanewdishwithspentmediumtoallowthemtogrowandexpand.ConcernsofSingleCellCloningHowdoyouknowwhenyouhaveatruesinglecellclone?Nomatterwhichmethodyouuse,therearestillsomedownsidestosinglecellcloning.Thecloningringmethoddoesnothaveabarrierandthereforecannotpreventcellsinonecolonyfrommigratingandgrowingwithanothercolony;thismayresultinnothavingatruesinglecellclone.However,thatiswhyIthinkmonitoringthecellstogenerateasinglecellcloneissoimportant.Asinglecellwillgrowmuchslowerthanifthereismorethan1cellaround.ThecellsIhaveworkedwithgenerallygrowfairlytightlytogetheriftheyarefromasinglecell,soItendtolookfortightlycompactedcolonies.SequencingresultsfromTOPOcloningexperimentshasalsohelpeddeterminewhetherIhaveasinglecellornot.IfIendupwithmultiplesequencesafterTOPOcloning,thisindicatesthatIactuallymayhavemorethan1clone.Although,tofigurethisout,IhavetorunalotofDNAsequencingreactions.Insomecases,IhavetakenaclonedcolonythatIsuspectmayactuallyhavemorethanoneclone,andperformedanotherroundofserialdilutions/cloningringtoensurethatitissinglecellcloned.BenefitstoSingleCellCloningItmaynotseemefficienttosinglecellcloneamutantcellline,asitistimeconsumingandcantakeabouttwomonthstocomplete,dependingonthegrowthofthecelllines.SometimesIhaveevenendedupwithasinglecellthathadtheGFPreporter,butturnsouttohaveaWTsequence.However,thatisexactlywhyIsinglecellsortafteratransfection:thepotentialpresenceofaWTsequencewouldseverelyconfoundmydata.Notonlythat,butIwouldbeafraidthattheWTcouldpossiblyevenoutcompetethegrowthofamutantcloneovertime,whichwouldalsonotbegood.Thus,Ithinkitisimportanttoisolatesinglecellclonesafterengineeringmutations,especiallywithsomethinglikeCRISPR/Cas9thatcangenerateheterogeneousmutants.Pleaseletmeknowhowthesetechniquesworkedoutforyou,orifyouhaveadditionalmethodsforsortingviablesinglecells.Sharethistoyournetwork:TwitterFacebookLinkedInWrittenbyChelseyKlineImageCredit:AlexandraERust1CommentAnthonyCheongonJune7,2019at12:59amIfyouhavesuspensioncellyoucanalwaysFACSsort,andusingaverysmallculturevesselhelps.Igenerate100%pureclonein25daysfromtransfection.LogintoReplyLeaveaCommentCancelReplyYoumustbeloggedintopostacomment.ThissiteusesAkismettoreducespam.Learnhowyourcommentdataisprocessed.ScrollToTop