Transgenic and Knockout Mice PROTOCOL相关交流-蚂蚁淘在线

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,meltat55^oCfor5min(flickingthetubeatleastevery1and1/2minutes).
Then,spinoutthesolutionfor30",wash2XwithNEWwash,spinning30"eachtime,drythefilterbyspinningfor1".
Elutethefragmentbyusing10-20µlofelutionbuffer,incubatingat37^oCfor5min(flickthetubeatleast2Xinthatperiod),spinningdown,andthenrepeatingtogetanyremainingDNA.
PrecipitatetheDNAovernight,using3MTrispH7.4orammoniumacetateasthesalt.If3MTrisisused,incubateovernightat-20^oC.Ifammoniumacetateisused,incubateovernightatroomtemp.
Then,do2X70%EtOHwashes.
ResUSPendinwhatevervolumelookslikeisappropriate.

Youcandialyzeovernightagainst2litersofinjectionbufferatthispoint,butit"sreallynotnecessary.Thismethodofpreppingfragmentsisprovingtobeareallyreliableandcleanmethod.Ihaveuseditonpiecesaslargeas18kB(that"tthereasonIwenttospincolumnsinthefirstplace).ThespincolumnsaresimplyaGeneCleankitwithafiltertocatchanyresidualglassbeads.TheliteraturethattheysendsaysthatthecolumnsaregoodforDNAfrom.2-200kB.

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PronuclearInjections:EggProductionforinjectionsToobtainalargequantitiy(>250)ofeggsforinjection,sexuallyimmatureFVB/Nfemales(4-5weeksofage)aresuperovulatedbyusingconsecutivePMSandHCGhormoneinjections.FemalesarematedtostudmalesimmediatelyfollowingtheHCGinjection.HarvestingtheeggsEggsareharvestedthenextdayfromtheampullaoftheoviductofthematedfemales.Eggsaretreatedwithhyaluronidasetoremovenursecells,andarethenwashedthroughseveraldishesofM2media.FertilizedeggsarethenstoredinM16mediaat37^oCandin5%CO2untilinjection.Injectingtheeggs30-50eggsareremovedfromtheincubatoratatimeforinjection.EacheggisindividuallyinjectedwiththeDNAfragmentofthedayunderhighmagnification.Wheneachegginthatgrouphasbeeninjected,alltheeggsarereturnedtotheincubator.Thisprocedureisrepeateduntilalleggshavebeeninjected.Attheendoftheinjectionperiod,eggswhichhavenotsurvivedinjectionareremovedfromeachgroup.ImplantingtheeggsInjectedeggsarethenimplantedingroupsof10-15bilaterallyintotheoviductofpseudopregnantfemales(femaleswhichhavebeenmatedtovasectomizedmales).Theanimalsareallowedtorecoverfromanaesthesiaonawarmingplate,andthenreturnedtotheanimalroom.Theyarekeptundersterileconditionsthroughouttheirpregnancy.

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MorulaAggregation:ThistechniquehasbeenbeautifullydescribedbyAndrasNagy(oneofthedevelopersofthetechnique)inhiswebpage.Clickonhisnametogothere.

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PCRforDeterminationofTransgenicMiceThisprotocolwasdevelopedbyJonNeumannattheUniversityofCincinnati

Earclipthemouse.Takethetissueandputintoamicrocentrifugetube.Cleanoffclipperscarefullybetweenanimals.
Digesteachclipin100µlof1XPCRbufferwithaddeddetergents(0.45%NP40,0.45%TWEEN20)and10µlProteinaseK(10mg/ml)@60^oCfor2hrs.toovernight.Ifusingashortincubationtime,vortexseveraltimesduringtheincubation.
DenaturetheProteinaseKbyboilingfor15min.(DONOTBOILFORANYLESSTHAN10min.;Reboilpriortoanysubsequentanalysis).Coolonicefor5minutes.
Aliquot18µlofPCRreactionbufferintoaPCRtube
PCRReactionBuffer
- 1XPCRbuffer
- 2.5mMMgCl2
- 200µMdNTPs
- 1µMeachprimer
- 1UnitTaqPolymerase
Add2µlofeardigesttothetube;mixbypipeting.
Overlaywith30-40µllightmineraloil(ifnotusinghottopPCRmachine)
Putintocyclerandrunthefollowingprogram:
- Hold@94^oCfor4.5min.
- 30XStepcyclesof:94^oCfor30sec.
- requiredannealingtemp.for20sec.(variesaccordingtoG/Ccontentofprimers)
- 72^oCfor1min.
- Hold@4^oCuntilreadytoanalyze.
Add2µlofagarosedyemixtoeachtube,andloadallontoa1.5-2%agarosegel.

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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).Airdryanddissolvein200µlTE
Use2µlofDNAsolutionforestimationofconcentration.

RestrictiondigestsofgenomicDNA:

-use10µgofDNAinatotalvolumeof300µl.
-allowwater,bufferandDNAtosittogether10-15minutesat37^oCpriortoaddingenzyme.
-digesttheDNA>7hours
-precipitatetheDNAwith2volumesofEtOH(besttodoovernight)
-spin,thenwashwith70%EtOH
-dry,thenresuspendin18µlofwater
-heattheDNAat65^oCforabout10"priortoloADIngonthegel
-add2µlofgelloadingdye
-runthegelslowly(about4V/cm)

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ObtainingaUniversityApprovedAnimalProtocolPriortoperforminganytransgenicwork,werequiretheinvestigatortohaveanIACUCapprovedanimalprotocol.InformationregardingtheproceduresforobtainingtheprotocolcanbeaccessedusingtheULARwebpage--justclickonthehighlightedULAR.

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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.
- 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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