ThepurityofthetransgeneDNAusedformicroinjectioniscriticalforthesuccessfulproductionoftransgenicfoundermice.DNAimpuritiesintheformofbacterialendotoxinsororganiccontaminantscandecreasetheintegrationefficiencyoftransgeneDNAortheviABIlityofmicroinjectedembryos.

AtransgeneconstructpreparedfromgenomicDNAispreferentialtoonepreparedfromaCDNAclone.WhiletherearesomereportsofcDNAtransgeneexpressionintheliterature,intronsintheDNAincreasethelikelihoodoftransgeneexpression.Plasmidvectorsequencesshouldalsobecleavedawayfromthetransgeneconstructtobeusedformicroinjection.Thepresenceofplasmidsequencesinterfereswithtransgeneexpression.ListedbelowareguidelinesforthepreparationofDNAformicroinjection.

UseplasmidDNAthathasbeenpurifiedonacesiumchloridegrADIenttoobtainDNAofhighestpurity.Digestapproximately20µgofplasmidDNAwitharestrictionenzymethatwillremovetheplasmidsequencesfromthetransgenicDNA.

RuntheDNAonanagarosegelandelutethebandcontainingthetransgenicDNAtobeinjected.Mostelutionmethodsworkfine.WehaveusedGeneCleanfromBio101.TheQiaExgelextractionkitfromQiagenwillalsowork.Phenolfreemethodsthatleavelittlesaltcontaminationaredesirable.

ResolubilizetheelutedDNAininjectionbuffer:

10mMTris,pH7.5

0.1mMEDTA,pH8.0

Whenpreparingtheinjectionbuffer,usedisposableplastic,freeoftracesofdetergents.Usereagentsdesignatedsolelyfortissuecultureworktoavoidtracecontaminants.Solutionsshouldbefilteredwitha0.2µmfiltertoremoveparticulateswhichmayclogtheinjectionneedle.ThepurityoftheDNAsampletobemicroinjectedmustbedocumentedbydigestingwithatleast2enzymesthatcutwithinthetransgeneandproducefragmentsofthepredictedsized.

SubmitageltoverifythepurityanddetermineconcentrationoftheDNA.Supply1-5µgofDNAadjustedtoafinalconcentrationof100ng/µl.WewilldilutetheDNAto1-3µg/mlformicroinjection.ToohighaDNAconcentrationwillcausedevelopmentalarrestofembryos.Toolowaconcentrationdecreasesthenumberoftransgenicfounders.DNAconcentrationisbestdeterminedbypreparingserialdilutionsoflamBDaDNAofknownconcentration(25,50,100,200,400ngoflambdaDNA)andusingthemasstandards.RunthelambdaDNAsandtheDNAtobemicroinjectedonageltodeterminetheDNAconcentrationofthedilutedDNAformicroinjection.Wewillalsoverifytheconcentrationbyfluorimetry.

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