Inroduction

Single-cellelectroporation(SCE)isatechniquewehavedevelopedtodelivergenesintoindividualcellswithinintacttissues,althoughitmayalsobeapplicabletocellsindispersecultures.TargetingtranscriptiontoindividualcellsisachievedbyrestrictingboththeDNAandtheelectricfieldrequiredforelectroporationtothe0.6-1µmtipofaglassmicroPipette.Thistechniqueisrelativelyeasytosetupandperform,canyieldhightransfectionrates,andrequiresrelativelyinexpensive,commonlaboratoryequipment.ApaperofthistechniqueispublishedintheMarchissueofNeuron,2001.

Thistechniqueutilizeselectroporationinwhichabriefexternalhighvoltagepulseinducessufficienttransmembranepotentialtodisrupttheelectrostaticforcesmaintaininglipidbilayerstructure,causingthetemporarilyformationofsmallporesinthecellmembrane.DNAandotherchargedmoleculesarethenelectrophoreticallytransferredintothecellthroughthesepores.Followingpulsetermination,poresresealover10sto100sofms.

SCEisapowerfultransfectionmethodsinceitreADIlyallowsthedeliveryofmultiplegeneseachcarriedbyindependentplasmidsintoasinglecell.Inaddition,SCEcanbeusedtotransfermacromoleculesbesidesDNAintocells,includingRNA,proteins,dyesanddrugs.Whilechargedmoleculesareactivelyelectrophoresedintocellswithhigherefficiency,nonchargedmoleculescanalsodiffusefromthemicropipetteintocellsthroughthepores.

TheequipmentrequiredforSCEisrelativelyinexpensiveandcommontomanyneurosciencelaboratories.SCEdoesnotrequirethepurchaseofmoreexpensivecommercialelectroporators.Ifyoudonothaveanelectricalstimulator,wearecurrentlydevelopinganinexpensivedeviceforgeneratingtheelectricalstimulineededforthistechnique.PleasecontactKurtHaasifyouwouldbeinterestedinsuchadevice.

ThemethoddescribedherehasbeensuccessfulfortransfectingindividualcellswithintheXenopustadpolebraininvivoandtherathippocampalorganotypicsliceculture.Modificationsofmicropipetteshape,electricalstimulationparameters,andmethodsoflocatingtargetcellsmaybenecessaryforotherpreparations.

Microscope:dissectingscope,oruprightwithlongworkingdistance,lowpower(~20X)objective

Voltagestimulator:GrassSD9Stimulator(Grass-Telefactor,WestWarwick,RI)**wearecurrentlydesigninganinexpensivevoltagestimulatorspecificallyforthistechnique.ContactKurtHaasforfurtherinformation

Oscilloscope:optional,butaidsinmonitoringpulseshapeandcircuitintegrityandelectroderesistance

Pipettepuller:P-87MicropipettePuller(SutterInstrumentCompany,CA)

Micropipetteholder:mustallowsliverwiretoextendfrombackofmicropipette

Manipulator:coarse,orcombinedcoarseandfinedependingonpreparation.

DNA:purifiedplasmidDNA

pipetteglass:glasscapillarytubing.borosilicate-standardwallwithfilament.outerdiameter=1.5mm,insidediameter=0.86mm.WarnerInstrumentCorp.

silverwire:0.25mmdiameter,toslideintomicropipette,andtouseasanexternalground

leads:toconnectmicropipettesilverwireandgroundsilverwiretovoltagestimulator

Micropipettes:Glassmicropipettesmustbecustomizedforeachpreparation.Wepullglasscapillarytubing(withfilament)usingaP-87MicropipettePuller.Ingeneral,apatch-clamptypeelectrodemaybesufficient.Thetipsizeshouldbearound0.6-1µmandhavearesistanceabout10Mwhenfilledwithstandardintracellularrecordingsolution.Shankdimensionscanvarydependingonthetissueusedandmustbalancerequirementsforpreventingthepipettefrombreaking(widershank)andreducingtissuedamagefromthepipetteinsertion(thinnershank).

DNAsolution:Genesofinterestmustbeplacedintoexpressionvectorscontainingpromotersappropriatefortissuetype.WepurifyourplasmidDNAusingPromegaWizardPlusMidiPrepsDNApurificationsystem(Promega,Madison,WI).WedilutepurifiedDNAto0.2-1µg/µlandfillthemicropipettetipwith0.6-1µl.EfficiencyofSCEwasnotnoticeablyeffectedbytheioniccompositionoftheresUSPensionsolution(2mMCaCl2,20-200mMNaCl,oronlydH2O)orDNAconcentrationsrangingfrom0.1to5µg/µl.DNAsolutionwasintroducedintothemicropipetteusingeithera1-10µmEppindorfPipettortip,ora1ccplasticinsulinsyringethathadbeenmeltedoverflameandpulledtoalongfinetip.

Circuitsetup:Athinsilverwire(diameter0.25mm)isinsertedintothemicropipettetouchingtheDNAsolutionatthetip.Themicropipetteisattachedtoacoarsemanipulatorwithapipetteholder.Asecondsilverwireisplacedindirectelectricalcontactwiththepreparation.ForSCEintadpoles,thegroundwireisplacednear(approximately1cm)thetadpoleunderaKimwipemoistenedwithsaline.Forhippocampalcultures,thegroundwireisplacedintheculturemedia.Thepositionofthegroundelectrodeisnotimportantaslongasitisincontactviaconductivesolutionwiththepreparation.

FortransferofnegativelychargedDNAintocells,thesilverwireinthemicropipetteisconnectedtothenegativeterminalofaSD9Grassvoltagestimulator.Thegroundsilverwireisconnectedtothepositiveterminalofthestimulator.

Microscope:Thetissue(here,eitherintacttadpole,orrathippocampalsliceculture)isplacedunderadissectingmicroscopeoranuprightOlympusBX50microscopeequippedwitha20Xlongworkingdistanceobjective.

Usingvisualguidanceatlowmagnification,thetipoftheDNA-filledmicropipettewasinsertedintothetissueinaregioncontainingdensecellbodies.Itwasnotnecessarytodirectlyvisualizethemicropipettetiporthecellbeingtransfected.Thehighdensityofcellbodiesinourtwopreparations(thecellbodyregionsoftheoptictectumoftheXenopustadpolebrain,andoftheCA1andCA3regionsoftherathippocampalslice)madeitlikelythattheelectrodetipwouldbeinclosecontactwithacellsomata.Inpreparationswithlessdensecellbodiesthisblindtechniquemayyieldlowtransfectionefficiencies.Inthesecases,itmaybebeneficialtomonitortipcontactwithcellseitherbydirectvisualization,orbyrecordingtheelectricalresistancechangesatthemicropipettetip.Directvisualizationmayalsobenecessaryifonerequirestargetingtoaspecificcell.

Stimulationparameters:WehavefoundthatawiderangeofelectricalstimulibetweenthemicropipetteandtheexternalgroundcanbeusedfortransfectionbySCE.WetestedsquarepulsesgeneratedbytheGrassSD9stimulatorandpulseswhichweremodulatedbyacapacitancecircuittoproduceasharphigh-voltagepeakfollowedbyanexponentialdecay.Wealsotestedtrainsofsquarepulses.

Transfectionofneuronsinthetadpolebrainwashighwithexponentialdecaypulseswithpeakvoltagesof20Vandt=70ms.Slightlyhighertransfectionefficiencywasachievedwith0.5-1strainsof1mslongsquarepulsesat50Vand200Hz.Wefoundthat5repeatedpulsesortrainsofpulsesalsoincreasedtransfectionefficiency.Itisusefultomonitortheelectricalpulsedeliveredtothepreparationwithanoscilloscope.Thiscantellwhetherthemicropipettehascloggedandhastobereplaced.Cloggingcanoftenbealleviatedbyapplyingbriefpulseswithalternatingpolarity.Ingeneral,thesamemicropipettecanbeusedatmanysites,allowingrapidinsertionandstimulationfollowedbyremovalandreinsertionatanothersite.Wefindthatmultiplerapidstimulationseffectivelycompensateforoccasionalincorrectmicropipetteplacementsduetoblindinsertiontoyieldadequatelyhightransfectionefficiencies.

Detectingtransfectedcells:WecommonlytesttransfectionsuccesswiththeClontech(ClontechLaboratories,PaloAlto,CA)plasmidpEGFP,whichdrivesgreenfluorescentproteinexpression(GFP)withastrongCMVpromoter.SinglecellstransfectedwithpEGFPexpressedbrightGFPwithin12hafterelectroporation,detectablebyepifluorescence.WerecommendfirsttestingSCEwithfluorescentdextrans(Molecularprobes,Eugene,OR),whichallowdirectvisualization,usingepifluorescence,ofdextransfillingcells.DuetotherelativelysmallsizeofdextranscomparedtoplasmidDNA,theelectricalstimulirequiredforSCEoffluorescentdextransismuchlessthanforDNA.

MethodforBulkTissueElectroporationLisaFoa,ClineLab

Wehaveadaptedelectroporationfortransferofmacromolecules,includingDNAtothetadpolebraininvivo.ElectroporationpermitsonetotargetDNAtransfectiontoselectedregionsononesideofthebrain,inadditiontowidespreadtransfectiondemonstratedbyothersintheirsystems.Electroporationalsoofferscontroloverthenumberofcellstransfected(Fig.2).TheequipmentandmaterialsrequiredforbrainelectroporationaresimilartoSCE.Wehavetestedarangeofconditionsandparameters.Outlinedbelowaretheelectroporationconditionsthatworkwellinstage44-48tadpolebraininvivo.

Microscope:Dissectingscope

Capacitor:Custommade

Voltagestimulator:GrassSD9stimulator(seeSCEnotesabove)

Oscilloscope:Optional(seeSCEnotesabove)

Picospritzer:PicospritzerII(GeneralValveCorporation)

Pipettepuller:P-87MicropipettePuller(SutterInstrumentCo.CA)

Micropipetteholder:Mustpermitpressureinjectionfrompicospritzer

Platinumelectrodes:custommade,platinumplateelectrodesapprox1x2mmsolderedtoelectricleads(forwiringtostimulatorandcapacitor)andmountedonarodforusewithamicromanipulator.

Micromanipulators:TwocoarseX,Y,Zmanipulators.Onetoholdthepressureinjectionpipette,onetoholdtheplatinumelectrodes.

Materials

DNA:0.2-2.0µg/µlpurifiedplasmidDNA

Pipetteglass:glasscapillarytubing,boroscilicate,standardwallwithfilament(WorldPrecisionInstrumentsInc.)Electrodetipdiameterwilldependontheapplication.

Methods

Micropipettes:AmicropipetteandPicospritzerareusedtopressureinjectDNAintothebrainventricle.Theshapeandsizeofthepipettetipisnotcritical,butitmustbesharpenoughtoeasilyPiercethetissue,andlargeenoughtoquicklydelivertheDNA.WeusethePicospritzerIItodeliver75-125nlDNAsolutiondirectlyintothetadpolebrainventricle.Thesamepipetteisusedformultipleanimals.

DNAsolution:Wetestedarangeofplasmidconcentrations(usingClontechpEGFP)andfoundthatconcentrationsrangingbetween0.2-2.0µg/µlyieldcomparablenumbersoffluorescentcells,withsimilarintensityofGFPfluorescence.DNAcanbedilutedindH2O,bufferedsaline,or2mMCaCl2.TheDNAsolutionwascoloredwith0.01%fastgreenasavisualaidforfillingthebrainventricle.Forco-electroporationoftwoplasmids,wemixplasmidsinaratioof1:1.Thistypicallygivesaco-transfectionrateof70%±10%(determinedforthesimultaneouselectroporationofofpEGFPandpDsRed).

Setup:Adissectingmicroscopewithgoodopticsissufficient.Themicromanipulatorsareplacedeithersideofthestage.Onemanipulatorholdsthemicropipette,connectedtothePicospritzer.Theothermanipulatorholdstheplatinumelectrodesconnectedtothecapacitorandstimulator.

Procedure:Theanesthetizedtadpoleisplacedonamoistenedkimwipeonthecenterofthemicroscopestage.ThemicropipettecontainingDNAisinsertedintotheventricleofthetadpolebrain,andtheDNAispressureinjectedintotheventricle.Forwidespreadelectroporation,DNAisinjectedtofilltheentirebrainventricle.Fortargetedelectroporationofaspecificbrainregion,aconcentratedbolusofDNAshouldbeinjectedascloseaspossIBLetotheregionofinterest.Themicropipetteisremoved,andtheplatinumelectrodesareimmediatelyloweredtocontactthetadpole"sskin,spanningthebrainregionofinterest(seeFig.2).2-7voltagepulsesaredelivered(dependingondesiredleveloftransfection).Effervescentbubblesareproducedattheelectrodetipswheretheycontacttheskin.Thelevelofeffervescenceisagoodindicatorofwhetheryouhaveachievedelectroporationvselectrocution.Thereshouldbenumeroussmallbubblesalongtheelectrodetips.Ifthebubblesarelargeandbubblingover,thevoltageistoolargeandtheanimalwilldie.Anothervisualcueistheamountofshockthetadpoledisplays.Thetadpoleeyesusuallyflickinresponsetotheelectroporation.Ifthewholebodyjolts,thevoltageistoolarge.Afterelectroporation,thetadpoleisquicklyreturnedtorearingsolution,whereitusuallyrecoverswithin10minutes.

TheDNAconstructscanbetargetedtojustonesideofthebrain,orifdesired,thewholebraincanbetransfected.Thisisachievedbyregulatingthevoltagepolarity.Ifonlyonesideofthebrainistobetransfected,thepolaritysettingonthestimulatorissetsothenegativelychargedDNAmovestowardsthepositiveelectrode.Ifbothsidesofthebrainaretobetransfected,thepolaritymustbeswitchedwhilethevoltagepulsesarebeingdelivered.

Stimulationparameters:Dependingonthenumberoftransfectioncellsdesired,2-7pulsesof30-50Vwithanexponentialdecayof70msareoptimal.Totransfectfewercells,reducethenumbersofpulses.

Detectingtransfectedcells:TransfectedcellsexpressingGFParedetectedbystandardfluorescencemicroscopy.

Troubleshooting:Weoccasionallyseesomebleedinginthebrainventricle24hrsafterelectroporation.Thisusuallyclearsupby48hrs.Propidiumiodidestainingindicatedthatelectroporationdoesnotcauseanincreaseincelldeath.Forgoodchargeconduction:-Ensurethatthespecimenremainsmoist-Theplatinumelectrodesmustbecleanedregularly.

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