Fordirectgenetransferoftibialisanterior(TA)muscleinmice:　

Itisoptimaltouse6-8weekoldmice(weight19-21gm).FemalesgivebetterimmuneresponsesforthehepatitisBsurfaceantigen,andthismightbetrueforsomeotherantigens.Thechoiceofmousestrainwillalsodependontheantigen.

MiceshouldbeanaesthetizedsinceawakemicewillcontracttheirmusclesandsqueezetheDNAsolutionout.Weuseeither:-sodiumpentobarbitalanesthesia(75mg/kgIP).-halothaneinhaledanesthetic(e.g.MetofanefromPittman-Moore).

Afterthemiceareasleep,thehindlimbsareshavedtobetterrevealthetibialboneandtheaccesstotheTAmuscle.Shavingofthelimbsallowsmuchgreaterprecisionandthusreproducibilityfortheactualinjectionstep.

Inpreparationfortheintramuscularinjection,DNAisdissolvedinendotoxin-freeinjectablePBS(NOTTRIS-EDTA)andisbestat0.1-2mg/ml(dependingonhowimmunogenicyourproteinisandhowrapidaresponseyouwant).See"PlasmidDNAPreparation"protocolsfordetailedinformation.

EachTAmuscleisinjectedwith50µlofDNAsolution.

ToinjectplasmidDNAusea27GX3/4"(0.4x20mm)needleattachedtoa1mltuberculinsyringe.Apieceofpolyethylenetubing(PE20,ID=0.38mm)shouldbefitovertheneedlesuchthatonly2-3mmofneedleprotrudes(basicallyjustthebeveledportionshouldprotrude).FillthesyringewiththeDNAsolution,attachtheneedleandthenslowlyfilltheneedlesothatnoairbubblesaretrapped.Theproblemofdeadvolumeissimplifiedusinganinsulinsyringe(seebelow).

Alternatively,useaU-100insulinsyringe(1ccor3/10cc)whichcomeswithapre-attached29G1/2needle.Polyethylenetubingisusedinthesamewayasdescribedabove.

Injectthroughtheskin-thetipoftheneedleshouldbeabout3mmlateraltotheanteriortibialtuberosity(thisisabouthalfwaybetweenthekneeandtheankle),keepingtheneedlealmostperpendiculartothetibia.Oncetheneedleisinplace(pushinuntiltheendofthePEtubingrestsagainsttheskinwithabitofpressure),injectthe50µlslowly(overapproximately10sec),holdtheneedleinplaceforanother5-10sec,thenremovetheneedleslowly.Ifyouaccidentallypulltheneedleoutbeforeinjection,trytoreinsertitinthesamehole,otherwiseyouwillexperienceleakage.

ItisagoodideatopracticeinjectionswithIndianinkorsomeothercoloredsubstancetomakesureyouinjecttheTAandonlytheTA-agoodinjectionwillnotcoloranymusclesotherthentheTA.

VariationsThereislessvariABIlityofresultsiftheDNAisinjectedin25%(w/v)sucrose(thisisnottrueforcardiotoxinpretreatedmuscle).

Forhigherefficiencyofgenetransfer,theDNAcanbeinjectedintoregeneratingmuscle(see"CardiotoxinInjection"protocol).Thisisaccomplishedbyfirstinducingasinglecycleofmyofiberdegenerationandregenerationbyintramuscularinjectionofcardiotoxin(100µlof10µMinPBS,pH7.4).WeuseacardiotoxinpurifiedfromthevenomoftheNajanigricollissnake(Latoxan,Rosans,France).TheDNAcanbeinjected5to9dayslater(optimaltimeseemstobe5d).IfyouareinjectingDNAintoregeneratingmuscle,DONOTusesucroseasthemusclefibersaretoofragileforthismuchosmoticpressure.

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ReferencesDavis,H.L.,Michel,M.-L.&Whalen,R.G.(1993)DNA-basedimmunizationforhepatitisBinducescontinuoussecretionofantigenandhighlevelsofcirculatingantibody.HumanMolecularGenetics2:1847-1851.Davis,H.L.,Whalen,R.G.&Demeneix,B.A.(1993)Directgenetransferintoskeletalmuscleinvivo:factorsaffectingefficiencyoftransferandstabilityofexpression.HumanGeneTherapy4:151-159.Davis,H.L.,Demeneix,B.A.,Quantin,B.,Coulombe,J.&Whalen,R.G.(1993)PlasmidDNAissuperiortoviralvectorsfordirectgenetransferinadultmouseskeletalmuscle.HumanGeneTherapy4:733-740.Davis,H.L.,Michel,M.-L.,Mancini,M.,Schleef,M.&Whalen,R.G.(1994)Directgenetransferinskeletalmuscle:plasmidDNA-basedimmunizationagainstthehepatitisBvirussurfaceantigen.Vaccine12:1503-1509.　

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ProtocolforPretreatmentofMammalianSkeletalMusclewithCardiotoxin[Seereferencesbelow]

CARDIOTOXIN

Forinductionofmuscledegenerationandregenerationbyintramuscularinjection

SOURCE:

Latoxan*A.P.1724F-05150Rosans,FRANCETel:+(33)-4.92.66.60.64Fax:+(33)-4.92.66.63.40

"CardiotoxinpurifiedfromvenomofNajanigricollis",CatalogRefNo.L81021mg:620FF+26%VATplusShipping&Handling5mg:2480FF+26%VATplusShipping&Handling

*Latoxanisusedtodeliveringabroad,andtheywillshipbyexpresscourierifdesired.Alternatively,cardiotoxinfromSigmahasbeenreportedtowork.

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PROTOCOL

1.Dilutestockto10-5M(=10µM)

Cardiotoxin:MW6800(1mg=1.47x10-7moles)

startwith1mgoflyophilizedpowder(onevial)

dissolvein14.7mlsterilesaline(0.9%NaCl)

filtertosterilizeifdesired

aliquotintoEppendorftubesandstoreat-20degreesCentrigrade

2.InjectintotheTibialisanteriormuscleof>20gmouseasfollows:100µlof10µMsolutionperleg(or50-75µlfor15-20gmmouse).Using27gneedlewithcollartolimitpenetrationto2mm,injectviaanteriorsurfaceofmuscle.

3.InjectionofDNAisoptimalfivedayslater(butisstillokayuptoninedayslater).Injectinsamemanner,50µlpermuscle.ForDNAimmunization,weinjectDNAat1mg/mlinPBS.

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REFERENCE:

Davis,H.L.,Demeneix,B.A.,Quantin,B.,Coulombe,J.&Whalen,R.G.(1993)PlasmidDNAissuperiortoviralvectorsfordirectgenetransferinadultmouseskeletalmuscle.HumanGeneTherapy4:733-740.

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SEEALSO:

d"Albis,A.,Couteaux,R.,Janmot,C.,Roulet,A.&Mira,J.C.(1988)Regenerationaftercardiotoxininjuryofinnervatedanddenervatedslowandfastmusclesofmammals.EurJBiochem174:103-110.

Whalen,R.G.,Harris,J.B.,Butler-Browne,G.S.&Sesodia,S.(1990)Expressionofmyosinisoformsduringnotexin-inducedregenerationofratsoleusmuscles.DevBiol141:24-40.

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PreparationofPlasmidDNAbyAnionExchangeChromatographyforIntramuscularInjectionREAGENTS:

Qiagenanionexchangecolumns

1XPBS:sterileandendotoxinfreefromSigma(Ref.D-5527).100XTE:sterileandendotoxinfreefromSigma.(Ref.T-9285)

GENERALCOMMENTS:

UseLBmediaandnot"TerrificBroth";Userecommendedbacterialstrains(e.g.,DH5a)DonotoverloadtheQiagencolumn(e.g.,thematerialfrom500mlofLB-grownculturegoesontooneMegacolumn);UsethebuffersprovidedbyQiageninthekit.

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PROTOCOL

1.DNAiselutedfromaQiagencolumn(theprocedureiscarriedoutfollowingpreciselytheQiagenprotocolANDusingthebuffersprovidedbyQiagen).Afterisopropanolprecipitation,theethanolwash,andvacuumdrying,theDNAistakenupinstandard1XTE(Sigma)Thisisconvenientlyleftovernighttoredissolve,althoughitwilldissolvemorerapidlyifneeded.

1bis.DNAfromaCsClgrADIentisextensivelydialyzedversus1XTE.(N.B.Qiagen-preparedDNAisessentiallyasgoodasDNApreparedbytworoundsofCsClgradients.)

2.AddNaCltotheDNA-TEsolutiontoafinalconcentrationof0.1M.

3.Thenadd2volumesofabsoluteethanol.Precipitateat-20degreesCentrigradefor30minutesandrecoverpelletbycentrifugation.

4.Washoncewith70%ethanolinwater(noEDTA)andrecoverbycentrifugation.

4bis.IftheDNAistobesenttosomeone,senditinastheEtOHprecipitateunderthe70%EtOHsolution.

5.Drythepellet,andresUSPenditin1XsterilePBS(Sigma).FromthesizeoftheQiagencolumnused,onecanestimatetheamounttoDNAtoberecoveredandaddthePBStoobtainasolutionofabout1.5to2.0mg/ml.Atthispoint,itisbesttoleaveDNA-PBSovernightat4degreesCentrigradetodissolve.Itisalsoveryhelpfultohavetheprecipitateinaroundbottom15mltoincreasecontactbetweendriedDNAandPBS.Ifnecessary,DNA-PBScanbewarmedat37degreesCentrigradetospeedupdissolution.

6.ReadOD260and280;the260/280ratioshouldbe>1.7foragoodDNAprep.DilutetheDNAto1mg/ml(orappropriateconcentration).Ifnecessary,storeat-20degreesCentrigradeorcolder.

7.DNAisnowreadyforinjection.