HumanmicroRNA(hsa-mir)precursorsandapproximately100-bpupstreamanddownstreamflankinggenomicsequenceswerePCRamplifiedandclonedintoaself-inactivated(SIN)lentiviralvectortogeneratepLV-miRNAvectors.Thecloningsiteofpre-miRNAgenomicfragmentsiswithintheintronofhumanEF1apromoterregion.ThemiRNAofinterestcanbedeliveredintocellsbytransienttransfectionofthepLV-miRNAplasmidorlentiviraltransductionwhilethemiRNAlentiviralstockispreparedfromcotransfectingHEK293TcellswiththepLV-miRNAplasmidandlentiviralpackagingvectormix.
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