Over-expression of Oct4 and Nanog represses the differentiation of C2C12 myoblast cells

• Main Authors: I-Hsuan Lin, 林依璇
• Other Authors: 陳盛良
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/63711838342913822443

碩士 === 國立中央大學 === 生命科學研究所 === 95 === Embryonic stem (ES) cells were originally derived from the inner cell mass of blastocyst stage mouse embryos. Recent studies have shown that two transcription factors, Nanog and Oct3/4, are responsible for sustaining the pluripotency of stem cells. In this study, we have over-expressed Nanog and Oct4, either individually or simultaneously, in C2C12 cells by retrovirus infection. We observed that the terminal differentiation of C2C12 myoblasts was repressed by Nanog-Oct4 over-expression. After analyzing RT-PCR expression pattern, either confluence (CMB) or differentiation (DM) stage, we found that expression of Pax7 is down-regulated in Oct3/4 over-expressed cells, but not in other stable clones. In the CMB stage, MyoD, MNF, Pax3, CD34, and HoxC10 were similarly expressed among stable clones. And Myogenin only expressed in the control and Nanog stable clones. Myf5 and Mrf4 were down-regulated in the double expression clone. MEF2C expressed in all stable clones, except for C2C12 control. M-cadherin was down-regulated , but Stella was up-regulated in the Oct4 stable clone. In the DM5 stage, MyoD, Myogenin, Myf5, Mrf4, MEF2C, and M-cadherin were down-regulation compared with others stable clones. Pax3 was down-regulated only in Nanog stable clone. HoxC10 was down-regulated in Oct4 stable clone. Using flow cytometric, we observe each of stable clones the phase of G0/G1, S, and G2/M is about 55%, 15%, and 26% at the PMB stage. In the CMB stage, Nanog and Oct4 stable clones were different form C2C12 control. To examine if Pax7 is targeted by OCT4 directly. We have made three clones of Pax7 promoter- 2K, 3.8K and 5K. The Pax7 3.8K and 5K promoter regions were regulated by OCT4. One of the putative Oct3/4 sites（from -4716 to -4776）could be bound by Oct3/4, as demonstrated by EMSA.