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.pptVIP

role of Tet proteins in 5mC to 5hmC conversion ES-cell self-renewal and inner cell mass specification 《Nature》 Conclusion of the paper ①. Tet ｝co-effect Nanog expression ES cell self-renewed DNMT Tet enhanced Nanog’s expression DNMT inhibited Nanog’s expression Tet have a essential role in maintaining ES cell fate The outline Ⅰ.Tet’s activity 1.Tet protein’s activity 2.Over –expression of Tet1,2 5hmC（only in wild-type） 3.Verifying the vitro activity of Tet Ⅱ. Tet’s function 4.Knockdown of Tet1 cause morphological abnormality(Tet1’s function in maintaining) 5.Tet1’s lack reduce ES cell growth rate.(Some factors’ level down) 6.Tet1’s knockdown make several factors level-up,lead to the cells differentiation 7.Tet1 binding place in Nanog’s promoter 8.Tet1 can cause methylation 9.Tet1 Nanog ES cells Ⅲ.Tet1’s function on ICM 10.Knock down Tet1 or not cause different proteins’ expression in ICM How to testify the results 1.Tet protein’s activity ways: Make Tet protein over-expressed then observed the 5mc staining level result:Tet1,2 reduced the staining but Tet3 wild-type worked well but the mutants ones 2.Over –expression of Tet1,2 5hmC（only in wild-type） ways:Use immunostaining to find whether 5hm C generated when Tets worked result:Tet1,2 can converted 5mC to 5hmC;Tet3 only add the 5hmC’s level without reduce 5mC’s quantities 3.Verifying the vitro activity of Tet ways:TLC and end labeled result:The generation of 5hmc followed by the enhanced expression of Tet 4. Knockdown of Tet1 cause morphological abnormality(Tet1’s function in maintaining) ways:Real-time PCR 、 shRNA to knock the Tet down result:Tet1,2 expressed in ES cell,but not Tet3;Knockdown of Tet1,but not Tet2,3 caused morphological abnormality. 5.Tet1’s lack reduce ES c