| Summary: | 碩士 === 國立中興大學 === 畜產學系所 === 94 === Transcription factors, Oct4 and Nanog, are believed to play a major role in the maintenance of cell pluripotency. They exclusively express in the early stage embryos, embryonic stem (ES) cells and pluripotent cells. It has been shown that the regulatory regions of Oct4 possess different transcriptional activity in a developmental pluripotency-dependent manner. No expression of Oct4 and Nanog is found in the differentiated cells, but over expression induces differentiation in ES cells. Hence, the aims of this study were to clone the regulatory regions of Oct4 for the reporter system in the production of transgenic animals, and to construct Oct4 and Nanog expression vectors (pCMV-Oct4 and pCMV-Nanog) to investigate the functions of Oct4 and Nanog in the regulation of germ layer differentiation. In Experiment 1, the promoter and enhancer regions were cloned from mouse genomic DNA and constructed into pCX-EGFP, the transcriptional activity of regulatory regions was analyzed by the expression of EGFP. The results showed that the constructed Oct4 regulatory regions could transcribe more efficiently in ES cells than in the differentiated cells, such as embryonic fibroblast cells and colon carcinoma cells. Additionally, the enhancer region increased transcription activity of Oct4 promoter in ES cells. After injection of EGFP gene driven by Oct4 enhancer and promoter into the pronuclei of mouse zygotes, the expression of EGFP was restricted to the inner cell mass of the blastocysts. It suggested that the cloned Oct4 regulatory regions possess a cell type-specific activity. In Experiment 2, mouse ES and 3T3 cells were transfected with pCMV-Oct4. The expression of Oct4 was analyzed by RT-PCR, Western blotting and immunofluorescent staining. Results showed that Oct4 was re-expressed in 3T3, and the expressions of Oct4 downstream genes, Esrrb, Rif1 and REST, were also increased. However, no expression of germ layer-specific genes was detected in transfected ES cells. In Experiment 3, the expression profiles of Nanog and Nanog downstream genes in 3T3 and mouse ES cells after transfected with pCMV-Nanog were similar to the results shown in Experiment 2. In conclusion, the gene constructs produced in this study are functional and could be applied to study Oct4- and Nanog-regulated cell pluripotency.
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