胚胎电转protocol

InvivoelectroporationintheembryonicmousecentralnervoussystemTetsuichiroSaitoDepartmentofDevelopmentalBiology,GraduateSchoolofMedicineChibaUniversity,Chiba260-8670,Japan.CorrespondenceshouldbeaddressedtoT.S.([e-mail])Publishedonline9November2006;correctedonline7and29December2006(detailsonline);doi:10.1038/nprot.2006.276Thisprotocoldescribesabasicmethodforinvivoelectroporationinthenervoussystemofembryonicmice.DeliveryofelectricpulsesfollowingmicroinjectionofDNAintothebrainventricleorthespinalcordcentralcanalenablesefficienttransfectionofgenesintothenervoussystem.Transfectionisfacilitatedbyforceps-typeelectrodes,whichholdtheuterusand/ortheyolksaccontainingtheembryo.Morethantenembryosinasinglepregnantmousecanbeoperatedonwithin30min.Morethan90%ofoperatedembryossurviveandmorethan90%ofthesesurvivorsexpressthetransfectedgenesappropriately.Geneexpressioninneuronspersistsforalongtime,evenatpostnatalstages,afterelectroporation.Thus,thismethodcouldbeusedtoanalyzerolesofgenesnotonlyinembryonicdevelopmentbutalsoinhigherorderfunctionofthenervoussystem,suchaslearning.INTRODUCTIONAnalyzinggenefunctioninmiceThestudyofgenefunctionandnetworksactivityinthebraininvivoisakeyissue,andmanynovelgeneshavebeenidentifiedbygenomeprojects.Thereareseveralwaystomanipulategenesinmice1.Transgenicandgenetargetingtechniqueshavegeneratednumerousmouselinesinwhichalteredgenesarestablytransmittedtonextgeneration.Recombinantviruses,liposomes2andbiolisticgeneguns3havebeenusedtotransfectgenesintoinvivotissues.However,transfectionbyliposomesorbiolisticsismostlylimitedtotissuesadjacenttothevascularsystemandsuperficialtissuessuchastheskin.Constructionoftransgenicandgene-targetedmiceandrecombinantvirusesistime-consumingandlaborious.Moreover,itisnotalwayseasytoexpressageneatthetimeandplacerequired,asthenumberoftranscriptionalregulatorysequences,suchasenhancersandsilencers,thatareavailabletorestrictgeneexpres-sionspatiotemporallyisstilllimited.Thus,quickandeasymethodsofgenetransferinmicewillgreatlyfacilitateourunderstandingofgenefunctionandnetworksinvivo.Invivoelectroporation:anoverviewElectroporationhasbeenwidelyusedtointroduceDNAintovarioustypesofcellsandtissues:prokaryotic4andeukaryoticcells5,6,mousemuscle7,inovochickembryos8,9andinvitromouseembryos9.Inmostcases,electricpulsesaredeliveredinasolutioncontainingcells,orbyinsertingelectrodesintotissues.Previously,wehaveshownthatgenescanbesuccessfullytransfectedevenintocellsthatarenotintheproximityofelectrodesbydeliveringelectricpulsestothemouseembryofromoutsidetheuterus,andthatthetransfectedgenesareexpressedinarestrictedareaofthenervoussystem10.Thisinvivoelectroporationhasbeensuccessfullyappliedtoanalyzegenefunc-tion10–16andtranscriptionalregulation14,17.Inthismethod,DNAismicroinjectedintothebrainventricleorthespinalcordcentralcanaloftheembryo,andsquare-waveelectricpulsesaredeliveredwithforceps-typeelectrodes.Treatedembryossurviveintheuterus,arebornandrearedbytheirmother.Genesaretransfectedintocellsthatarelocatedadjacenttotheventricleorthecentralcanal10,11,18.Asmanyofthetransfectedcellsareneuronalprogenitors,theyandtheirdescendantneuronsexpressthetransfectedgenes.Toseetheembryomoreclearlyduringtheprocedure,theuterinewallcanbecutbeforeDNAinjection10.Operatedembryosthatareconnectedtotheplacentacansurviveintheyolksacinsidethepregnantmouse,andpupscanberecoveredbycesareansectionandrearedbyafostermother.Electroporationoutsidetheuterinewallisdescribedasexouteroelectroporation,whereasinuteroelectroporationdescribestheprocedurewhentheuterinewallisleftuncut.Invivoelectropor-ationdescribesbothinuteroandexouteroprocedures.Geneshavebeensuccessfullytransfectedtothetelencephalon10,13,15–19,dien-cephalon10,midbrain10,hindbrain20andspinalcord11,12,14.AdvantagesofinvivoelectroporationInvivoelectroporationhasthefollowingadvantageousfeatures:Theprocedureissimpleandquick.Invivoelectroporationforgenetransferintomorethantenembryoscanbecarriedoutwithin30min.Transfectionefficiencyishigh(seeTable1).However,despitethishighefficiency,cytotoxicityremainslow.Nosignificantincreaseincelldeathhasbeendetectedafterelectroporation(ref.18;TS,unpublishedresults).Multiplegenesondifferentplasmidscanbesimultaneouslytransfectedintothesamecells10,indicatingthatthismethodcanbeusedtoanalyzecombinedfunctionofgenes.Incontrast,transferofmultiplegenesisnoteasyforrecombinantviralsystems,becausethesizeofDNAthatcanbeincorporatedintoviralparticlesislimited.Plasmidsthatarelargerthan10kbcanbesuccessfullytrans-fected10,18.Transfectionisunidirectional.Onlythesideoftheventriclethatisclosesttotheanodeistransfected10,11.Thisfeatureisusefulforanalyzinggenefunction,asthenon-transfectedsideservesasanegativecontrolonthesamesection.Transfectionistemporallyrestricted.Expressionofatransfectedgeneislimitedtoparticulartypesofneuronsbecausegenerationofneuronaltypesisdependentonembryonicstage.Inthecerebralcortex,progenitorsthataretransfectedataparticularstagegiverisetoneuronsofspecificlayers10,18.Distinctgenescanbeexpressedbyneuronsofdifferentlayersbydoubleelectropo