Color-Switch,CREreporter stablecellline:
TheCREreportingcelllinesareusedtomonitororconfirmtheefficiencyofCRErecombinationinvivo.ItisagreatmethodandeasytooltoverifytheperformanceofyourCREenzyme(yourCREexpressionplasmids,orpre-madeCREexpressionlentivirus,orpurifiedCREenzyme)invivoconditions.ItisalsoacontroltesttoverifyyourCRE-loxPbasedsystem.TransformedfromHEK293cells, expressing“LoxP-GFP-stop-LoxP-RFP”cassetteunderCMVpromoter(seeColor-Switchcassettebelow).Celllinealsocontainsa NeomycinantibioticMarkerunderRSVpromoter.
ThecelllinedemonstratesstrongGFPfluorescentsignalinnormalcultureconditionastheconstitutiveCMVpromoterdrivestheGFPhighexpressiontothestopcodon.ThedownstreamRFPORFwasnotexpressedbecauseofthestopcodonaftertheGFPORF.OncetheCREproteinwaspresentinnuclear,theCREexcises/deletestheDNAfragmentbetweentwoloxPsites,whichremovesthestopcodon(seetheDNA structureschemeabove).Asaresult,theRFPORFisthenexpressedundertheCMVpromoter,andthecelllineswitchestoRFPfluorescent.TheRFPsignalcanbeeasilymonitoredviafluorescentcellsorting(fortheratiobetweenGFPandRFPcells),orvisualizedundermicroscope,ormeasuredthefluorescentintensitybyameterorreaderwithRFPfilterset.
SampleimagesofCRE-loxPrecombinationdetection:
Leftpanel/withoutCRE:CREreportercellline(Cat#:SC018-Bsd)wasculturedincompleted.ImagesweretakenundermicroscopewithGFPfilterset(Ex490nm/Em525nm)andRFPfilterset(Ex545nm/Em620nm).
Rightpanel/withCRE:CREreportercelllinewasculturedincompletedin24-wellplate.50ulofCREexpressionlentiviralparticle(Cat#:LVP339)wasaddedintothecellsinonewell.Imagesweretakenat~72hoursaftertheadditionofCREexpressionlentivirus.
PleaseclickhereforProductManual.
Soldat: 1vialx(2 x106cells)/vial
Cat#:SC018-Neo
