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1、上海交通大学硕士学位论文investigatetheregulatorymechanismofTFexpression.TheresultshowedthattheRPpromoterdirectedanefficienthTFgeneexpression,andtheadditionof5-aza-dCincreasedhTFexpressioninaproportionalmanner,indicatingthehTFexpressionwasregulatedbyamethylationmechanism.Theresultsalsoshowedthatthele
2、velsofhTFexpressiondifferedbetweenclones,suggestingthatTFexpressionwasalsoinfluencedbytheintegratedsites,copynumbersandsoon.Inordertoresearchtherelationshipbetweenmethylationandgeneexpressionatindividuallevel,wegeneratedanumberofhTFtransgenicmicebylentiviralvectormediation.Withreal-timeP
3、CRanalysisweidentifiedtheF1generationoftransgenicmicewithsinglecopyandsinglesitehTFintegration.hTFtranscrpitsineightkindsoftissues(heart,liver,spleen,lung,kidney,brain,testicle,muscle)ofthreeF1mice(F1-797,F1-949,F1-972)wereexamined.TheexpressionprofilesofhTFinthreemiceweresimilar.hTFwase
4、ffectivelyexpressedinliverandbrain,butlowerinmuscle.Furtheranalysisoftheexpressionandmethylaitoninliver,heartandmuscleindicatedconfirmedthat,thehighertheexpressionofhTF,thelowerthemethylationlevelinRPpromoterwas,andviceversa.TherelationshipbetweenpromotermethylationandhTF-V-上海交通大学硕士学位论文e
5、xpressioninliversofF1-797,F1-949andF1-972wasinvestigated.Theresultsshowedthattheexpressionlevelwasinverselycorrelationwithmethylationstatus.InhigherhTFexpressionmice(F1-797andF1-949),thepromotermethylationsituationinliverswaslow,whileinlowerhTFexpressionmouse(F1-972),themethylationsituat
6、ionwashigher.ThecontributionofeachCpGsitetothegeneexpressionwasstudied.TheresultsshowedthatthemethylationlevelsoftheCpGinRPtranscriptionfactorbindingsitePRI,RNApolymerasebindingsiteandKOZAKsequence,werelowerthantheaveragemethylationlevelwhenmorehTFexpressed.Thelowmethylationinthesesitesm
7、ayenhancethetranscriptionfactorbindingonthetargetsequenceandimprovethegeneexpression.ThisthesisprovesthattheexogenousgenehTFwasregulatedbymethylationmechanisminbothcellularandindividuallevel.Thestudygivesrisetothepossibilityandfeasibilitytodeveloptransgenicanimals/mammalb
