| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 94 === Embryonic stem cells are pluripotent progenitors for virtually all cell types in our body. Recent studies have shown that some transcription factors can sustain pluripotency of the stem cells. These regulators activate or repress patterns of gene expression that mediate phenotypic changes during stem cell differentiation. Nanog has recently been proposed to play a key role in maintaining stem cell pluripotency. Oct4 (Oct3), a member of the POU family of homeodomain proteins, can regulate the expression of their target genes through binding to an octameric sequence ATGCAAAT. Here, we try to explore whether over-expression of Nanog or Oct4 (Oct3) will re-establish the pluripotency of determined myoblasts and thus prevent its terminal differentiation? Using retrovirus carrying Nanog or Oct4 cDNA, we have engineered cell lines that can stably express Nanog or Oct4 or both of them. We further demonstrated that the number of the myotubes in Nanog over-expressed cell is about one fifth to that of retrovirus control infected cell. In the meantime, the number of myotubes is 59 in Nanog over-expressed cell and is 236 in retrovirus control infected cell. Morphologically, the Nanog overexpressed myotube is more matured than the retrovirus control infected cell. Our preliminary results show that Nanog actually influence the degree of differentiation not only in the number of myotube formation but also in the state of myotube maturation. Furthermore, we also found that expression of MyoD and Pax7 is downregulated in Oct4 overexpressed cells. In transient transfection reporter assays, Nanog functions as an activator, but Oct4 functions as a repressor in C2C12 cell line. We also provide evidence showing that Nanog can form homodimer complex and also interacts with Oct4 weakly. Based on the homeodomain, Nanog can be divided into three regions. In order to investigate the transactivation activity of Nanog N terminal domain, we used the well-established Gal-4 reporter system. We report here that N terminal domain of Nanog could perform different transactivation activity in different cell type under different promoter context. By using GST pull down experiment, we further demonstrated the relationship among Nanog, Oct4 and SMRT.
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