The Regulation Of OCT4 Expression By TPT1 Gene

In order to study the regulation of Oct4 by TPT1 gene , dual-luciferase reporter assay and real-time quantitative PCR were carried out using P19 cells as a model. And the results are as follows:1. We cloned mouse Oct4 promoter by the method of molecular cloning, and verified by DNA sequences.Then the Oct4 promoter was subcloned into pEGFP-1 to construct pOct4-EGFP. To determine the function of Oct4 promoter, the plasmid pOct4-EGFP was transfected into P19 EC cells and NIH3T3 fibroblasts. The Oct4 promoter directed EGFP expression was observed in P19 cells, but absent in 3T3 fibroblasts, indicating that the Oct4 construct has the tissue specificity. Finally, the Oct4 promoter was subcloned into pGL3-Basic to construct pOct4-luc.Meanwhile, the mouse TPT1 cDNA coding region was subcloned into pcDNA3-FLAG and pEGFP-C1 to construct pcDNA3-TPT1 and pEGFP-TPT1. pcDNA3-TPT1 and pEGFP-TPT1 were transiently transfected into P19 cells and conducted the western blot assay. The rusult demonstrated that the exogenous TPT1 gene was highly expressed in P19 cells.2. Using P19 cells as a model, through the Dual-Luciferase detection system, our results showed that along with TPT1 overexpression, Oct4 expression was significantly decreased .To further monitor TPT1 effect on suppressing Oct4 expression, the time-dependent experiments were carried out to discover the better interaction time between TPT1 and Oct4. Cell samples were collected at different time points, and were analyzed by luciferase assays. To examine the dose-dependent effects of TPT1 on Oct4 expression, the different amounts of pcDNA3-TPT1 plasmid were co-transfected with pOct4-Luc vector. At 0.5μg of pcDNA3-TPT1 treatment, 80% of Oct4 activity was reduced . These results indicated again that over expression of TPT1 in embryonic stem cells could suppress the expression of Oct4. To examine the endogenous expression of TPT1 in P19 EC cell, 3T3 fibroblast, C2C12 myoblast and J1 ES cell, quantitative PCR assays were carried out by the method of real-time RT-PCR. The results demonstrated that TPT1 mRNA was detected in whole four cell lines. However, the mRNA level of TPT1 was 2-4 folds lower in embryonic cell lines (P19 and J1) than that in somatic cell lines (3T3 and C2C12). Then, The exogenous TPT1 was transfected into P19 cells, and the mRNA level of TPT1 was determined. In the transfected cells, TPT1 level was 13 folds higher than un-transfected P19 cells. However, in TPT1 transfected P19 cells, the mRNA level of endogenous Oct4 was reduced 25 folds comparing with that of control cells. This observation indicated that over expression of TPT1 might control at the transcriptional level the suppression of Oct4 gene expression in embryonic cells.The mouse ES cells J1 were transfected by plasmid pcDNA3-TPT1 for research on the effect of growth of stem cell. Then, The alkaline phosphatase staining was done after 24 h. Compared with the control, the transfected with pcDNA3 -TPT1 of J1 cells were weakly positive from the color, and clones become loose from the morphological, which is a tendency to differentiate. Then, by building TPT1 and GFP fusion expression vector transfected P19 EC cell and NIH3T3 cells, initially identified TPT1 location in the cell is not the nucleus, which implies us that TPT1 indirectly inhibit the transcription of Oct4.