蛋白在es细胞未分化状态维持和内细胞团特化中的功能课件

roleofTetproteinsin5mCto5hmCconversionES cellself renewalandinnercellmassspecification Nature Conclusionofthepaper Tet co effectNanogexpressionEScellself renewedDNMTTetenhancedNanog sexpressionDNMTinhibitedNanog sexpression TethaveaessentialroleinmaintainingEScellfate Theoutline Tet sactivity1 Tetprotein sactivity2 Over expressionofTet1 25hmC onlyinwild type 3 VerifyingthevitroactivityofTet Tet sfunction 4 KnockdownofTet1causemorphologicalabnormality Tet1 sfunctioninmaintaining 5 Tet1 slackreduceEScellgrowthrate Somefactors leveldown 6 Tet1 sknockdownmakeseveralfactorslevel up leadtothecellsdifferentiation7 Tet1bindingplaceinNanog spromoter8 Tet1cancausemethylation9 Tet1NanogEScells Tet1 sfunctiononICM 10 KnockdownTet1ornotcausedifferentproteins expressioninICM Howtotestifytheresults 1 Tetprotein sactivityways MakeTetproteinover expressedthenobservedthe5mcstaininglevelresult Tet1 2reducedthestainingbutTet3wild typeworkedwellbutthemutantsones 2 Over expressionofTet1 25hmC onlyinwild type ways Useimmunostainingtofindwhether5hmCgeneratedwhenTetsworkedresult Tet1 2canconverted5mCto5hmC Tet3onlyaddthe5hmC slevelwithoutreduce5mC squantities 3 VerifyingthevitroactivityofTetways TLCandendlabeledresult Thegenerationof5hmcfollowedbytheenhancedexpressionofTet4 KnockdownofTet1causemorphologicalabnormality Tet1 sfunctioninmaintaining ways Real timePCR shRNAtoknocktheTetdownresult Tet1 2expressedinEScell butnotTet3 KnockdownofTet1 butnotTet2 3causedmorphologicalabnormality 5 Tet1 slackreduceEScellgrowthrate Somefactors leveldown ways MeasurethelevelofsomekeystemcellfactorsResult Nanog Oct4 Sox2 Q LIF slackmakeTetanditsmRNA Whydotheystudythese 6 Tet1 sknockdownmakeseveralfactorslevel up leadtothecellsdifferentiationways KnockdownTet1toobserveSSEA 1 schange StaningCdx2Hand1Gata6 result SSEA 1 Cdx2 Hand1 Gata6 7 Tet1bindingplaceinNanog spromoterways ChIP real timeqPCRresult Tet1bindedtosomeplacenearNanog spromoter 8 Tet1cancausemethylationways twogrouptocompare DNMTlackornot LetTet1down observedNanog sexpressionresult InnormalonesTet1 Nanog InDNMTdefectedonesTet1 Nanog slevelchangedalittleThatmeansNanogisTet1 sdirecttarget 9 Tet1NanogEScellsways KnockeddownTet1addNanog measuredthemorphologicalfeatureandCdx2 Gata6 squantitiesresult withoutTet1 onlywithNanogcanmakeEScellrenewedpartly 10 KnockdownTet1ornotcausedifferentproteins expressioninICMways RT qPCRinjectH2B MFPwithsiTetstaningOct4orCdx2 respectivelyresult Tet sknockdownbringOct4 Cdx2 sexpressionincreased DNMT3amethylationMutationby DNMT3aMutationsinAcuteMyeloidLeukemia Both5mCand5hmCcaninhibittheactivityoftranscription ThetransferpathwayTetTetdioxygenaseTDG5mC5hmC5caCC Howtoprovetheexistenceof5mCand5hmC SMRT single moleculereal timePCR Specificenzymeandsomerelativeantibodies Thesupplement 1 AIDisinvolvedintheprocedureofdemethylation4 Synonyms AICDA198AAlengthItcanmakeCytidine H2Otouridine NH3 2 Transcriptionalelongatorisrequiredforpaternalgenomedemethylationinzygotes 3 Some5hmC specificDNAglycosylasecanhelp5hmCtoC Nanog蛋白研究新进展来自于爱丁堡大学的IanChambers博士的研究小组 在未分化的干