The Expression Of Pkm2 During The Differentiation And The Construction Of Reporter System Of Pkm2

Objective:1、We aimed to examine the expression of pyruvate kinase in the ES cells,fibroblasts,tissues and mouse organs and explore the role of Pkm2 in ES cells and induced pluripotent stem cells.2、We wanted to examine the expression of Pkm2 during the reprogramming and the ES cells differentiation process.3、We used the CRISPR/Cas9 system to obtain ES cells stably expressing the red fluorescent protein under the pkm promoter.4、We aimed to employ the Piggy Bac system to explore the new regulatory protein that could influence the expression of pkm2, and then test the relationship between the new protein and Pkm2.Methods:1、We obtained the tissues and organs,the cells of fibroblasts,ES cells and the induced pluripotent stem cells from the mouse.2、We utilized the methods of the realtime q PCR to test the expression of Pkm2,Pkm1,Pklr in the different tissues,organs and cells.3、We explored the expression of pkm2 and the genes of Ptb、P53、Hif-1a、Hnrnp a1 in ES cells and in the reprogramming process. We also examined the differentiated gene such as Pax6,Foxa2 and Gata6, the gene such as Pkm2,Pkm1,the pluripotent genes such as Nanog and Oct4 and the genes of Ptb、P53、Hif-1a、Hnrnp a1 during the differentiation process.4、We designed the sg RNA and used the system of the CRISPR/Cas9 and transfected the plasmid into the ES cell.5、We got the cells with the red fluorescent protein and purified the cell lines indicating the expression of pkm.6、We tested the Piggy Bac system in 293 T cell lines.Results:1、Pkm2 expresses highly in the ES cells and the i PS cells,and Pkm2 plays an important role in reprogramming and in differentiation process.2、We got the ES cell lines with the red fluorescent protein indicating the expression of pkm.3、We tested the Piggy Bac system in 293 T cells.