- DNAPreparationformicroinjectionorelectroporation
- Pronuclearinjectiontoproducetransgenicmice
- MorulaAggregationtoproducechimericmice
Transgenicidentification
- PCRofDNAfromearcliptissue
- GenomicSouthernsofDNAfromtailclips
AnimalProtocols
- ObtainingaUniversityapprovedanimalprotocol
- Matingtransgenicmice
DNAPreparationforMicroinjectionorElectroporationThisprotocolwasdevelopedbyJanParker-ThornburgatTheOhioStateUniverstiy1)RuntheDNAdigestonanagarosegel:- Makea1%Seaplaque(orotherveryhighquality)Lowmeltagarosegelusing1XTAEpreparedaccordingtoManiatis(noEtBr).
- LoadthegelwithMarkersontheendseparatedfromthedigestofinterestby1lane.(loadtheDNAataconcentrationofnomorethan200ng/lane--otherwiseyouruntheriskoftrappingfragmentsthatyoudon"twantinthebandofinterest.)
- Runthegelslowly:30-40mAmps.
- Oncetheruniscompleted,stainthegelwithafairlylowconcentrationofEtBr(about1µg/ml)for10minkeepingthegelinthedark.
- Whenfinishedstaining,visualizegelusinglongwaveUV,andcutthebandofinterest.
2)PurifiyingtheDNAfragment.- Loadabout200-300µgofthegelcutontoaGeneCleanspincolumn(BIO101"SGeneCleanSpinkit)--usuallyafragmentwillrequire6-8columnstotakecareofallthegel.
- Priortoaddingthefragment-ladengelpieces,add500µlofglassmilk.
- Oncethegelisinthetube,meltat55oCfor5min(flickingthetubeatleastevery1and1/2minutes).
- Then,spinoutthesolutionfor30",wash2XwithNEWwash,spinning30"eachtime,drythefilterbyspinningfor1".
- Elutethefragmentbyusing10-20µlofelutionbuffer,incubatingat37oCfor5min(flickthetubeatleast2Xinthatperiod),spinningdown,andthenrepeatingtogetanyremainingDNA.
- PrecipitatetheDNAovernight,using3MTrispH7.4orammoniumacetateasthesalt.If3MTrisisused,incubateovernightat-20oC.Ifammoniumacetateisused,incubateovernightatroomtemp.
- Then,do2X70%EtOHwashes.
- ResUSPendinwhatevervolumelookslikeisappropriate.
Youcandialyzeovernightagainst2litersofinjectionbufferatthispoint,butit"sreallynotnecessary.Thismethodofpreppingfragmentsisprovingtobeareallyreliableandcleanmethod.Ihaveuseditonpiecesaslargeas18kB(that"tthereasonIwenttospincolumnsinthefirstplace).ThespincolumnsaresimplyaGeneCleankitwithafiltertocatchanyresidualglassbeads.TheliteraturethattheysendsaysthatthecolumnsaregoodforDNAfrom.2-200kB.BacktoPROTOCOLS
PronuclearInjections:EggProductionforinjectionsToobtainalargequantitiy(>250)ofeggsforinjection,sexuallyimmatureFVB/Nfemales(4-5weeksofage)aresuperovulatedbyusingconsecutivePMSandHCGhormoneinjections.FemalesarematedtostudmalesimmediatelyfollowingtheHCGinjection.HarvestingtheeggsEggsareharvestedthenextdayfromtheampullaoftheoviductofthematedfemales.Eggsaretreatedwithhyaluronidasetoremovenursecells,andarethenwashedthroughseveraldishesofM2media.FertilizedeggsarethenstoredinM16mediaat37oCandin5%CO2untilinjection.Injectingtheeggs30-50eggsareremovedfromtheincubatoratatimeforinjection.EacheggisindividuallyinjectedwiththeDNAfragmentofthedayunderhighmagnification.Wheneachegginthatgrouphasbeeninjected,alltheeggsarereturnedtotheincubator.Thisprocedureisrepeateduntilalleggshavebeeninjected.Attheendoftheinjectionperiod,eggswhichhavenotsurvivedinjectionareremovedfromeachgroup.ImplantingtheeggsInjectedeggsarethenimplantedingroupsof10-15bilaterallyintotheoviductofpseudopregnantfemales(femaleswhichhavebeenmatedtovasectomizedmales).Theanimalsareallowedtorecoverfromanaesthesiaonawarmingplate,andthenreturnedtotheanimalroom.Theyarekeptundersterileconditionsthroughouttheirpregnancy.BacktoPROTOCOLS
MorulaAggregation:ThistechniquehasbeenbeautifullydescribedbyAndrasNagy(oneofthedevelopersofthetechnique)inhiswebpage.Clickonhisnametogothere.BacktoPROTOCOLS
PCRforDeterminationofTransgenicMiceThisprotocolwasdevelopedbyJonNeumannattheUniversityofCincinnati- Earclipthemouse.Takethetissueandputintoamicrocentrifugetube.Cleanoffclipperscarefullybetweenanimals.
- Digesteachclipin100µlof1XPCRbufferwithaddeddetergents(0.45%NP40,0.45%TWEEN20)and10µlProteinaseK(10mg/ml)@60oCfor2hrs.toovernight.Ifusingashortincubationtime,vortexseveraltimesduringtheincubation.
- DenaturetheProteinaseKbyboilingfor15min.(DONOTBOILFORANYLESSTHAN10min.;Reboilpriortoanysubsequentanalysis).Coolonicefor5minutes.
- Aliquot18µlofPCRreactionbufferintoaPCRtube
- PCRReactionBuffer
- 1XPCRbuffer
- 2.5mMMgCl2
- 200µMdNTPs
- 1µMeachprimer
- 1UnitTaqPolymerase
Add2µlofeardigesttothetube;mixbypipeting.Overlaywith30-40µllightmineraloil(ifnotusinghottopPCRmachine)Putintocyclerandrunthefollowingprogram:- Hold@94oCfor4.5min.
- 30XStepcyclesof:94oCfor30sec.
- requiredannealingtemp.for20sec.(variesaccordingtoG/Ccontentofprimers)
- 72oCfor1min.
- Hold@4oCuntilreadytoanalyze.
Add2µlofagarosedyemixtoeachtube,andloadallontoa1.5-2%agarosegel.BacktoPROTOCOLS
TailDNAPrepsforGenomicSouthernsThisprotocolwasdevelopedbyArunSubramaniamfromProctorandGambleDigestionofthetailclip:- Cutabout50-100mgsoftailintoanEppendorftubecontaining500µloftailprepsolution:
- 50mMTrispH8.0
- 100mMEDTA
- 100mMNaCl
- 1%SDS
- 750µgProteaseK/ml
Incubateat600Covernight(oruntildigestioniscomplete)Add0.5mlphenol,mixwell,spinfor10",10,000rpmatroomtemperature.Removeaqueousphase.Totheaqueousphase,add0.5mlphenol/chloroform,mixwell,thenspinfor10",10,000rpm,roomtemperature.Removeaqueousphase.Totheaqueousphase,add0.5mlchloroform,mixwell,spinfor5",10,000rpm,roomtemperature.Removeaqueousphase.Tothatphase,add0.5mlcold95%EtOH.Mixwell.Youwillseeawhiteballofpptform.ThatisthegenomicDNA.SpooloutDNAwithPipettetip.(Youactuallydigthisoutwiththetip,ratherthanspoolingit).RinseDNAin70%EtOH(Cold),andthen95%EtOH(Cold).Airdryanddissolvein200µlTEUse2µlofDNAsolutionforestimationofconcentration.RestrictiondigestsofgenomicDNA:- -use10µgofDNAinatotalvolumeof300µl.
- -allowwater,bufferandDNAtosittogether10-15minutesat37oCpriortoaddingenzyme.
- -digesttheDNA>7hours
- -precipitatetheDNAwith2volumesofEtOH(besttodoovernight)
- -spin,thenwashwith70%EtOH
- -dry,thenresuspendin18µlofwater
- -heattheDNAat65oCforabout10"priortoloADIngonthegel
- -add2µlofgelloadingdye
- -runthegelslowly(about4V/cm)
BacktoPROTOCOLS
ObtainingaUniversityApprovedAnimalProtocolPriortoperforminganytransgenicwork,werequiretheinvestigatortohaveanIACUCapprovedanimalprotocol.InformationregardingtheproceduresforobtainingtheprotocolcanbeaccessedusingtheULARwebpage--justclickonthehighlightedULAR.BacktoPROTOCOLS
MatingProtocol- Determinetheageofyourmice.Micewillusuallynotbreediftheyareyoungerthan4weeksold.Similarly,micewhohavebeenhousedaloneorinpairs(withthesamesex)willusuallynotbreediftheyareolderthan6-8monthsold.Onoccasion,oldermicewillbreedsuccessfully,butthefemalescanalsohavesignificantproblemswithdeliveringlivepups,andnursingthemappropriately.Asasuggestion,ifhousingmicelongtermwherethereisachancethatyoumayneedtobreedtheminthefuture(suchastokeepthelinegoing),youwillwanttobreedthematleastoncepriorto6monthsofage.Thepupsfromthisbreedingmaybesacrificedifnotneeded.Thiswillhelpwhenfuturebreedingisattempted.Remember,oldmicearealotlikeoldpeople.Astheyage,thequalityandquantityofbothspermandeggsdecrease.Notonlyaretherephysiologicalchanges,buttherearetemperamentchangesaswell.Theycanbecomegrumpyoldmice.
*Minimumbreedingage:- males:35-42days
- females:21days*Maximumageforfirstbreeding:
- malesandfemales:6months
Setupbreedingsonlywhenyouknowthatthemicewillbesupervisedcarefullyduringthenextday.Onoccasion,themicewillfightafterbreeding,andcaninflictseriouswoundsthatcouldbefatalwithoutpromptattention.Thus,miceshouldnotbebredonFridaysorSaturdaysoronSundayonalongweekend.Tobreed,putthefemaleintothemale"scage.Reversingthisordercanresultinthemalekillingthefemale(or,onrareoccasions,thefemalekillingthemale).IfitisnotpossIBLetoputthefemaleintothemale"scage,useacleancage,andputthemaleinthecagefirst.Ifthiscanbedoneoneweekinadvanceoftheanticipatedmating,thiswillallowthemaletomarkhisterritory,andthepheremoneleveltorise,whichwillaidinthebreedingprocess.
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