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標題: 多能性相關基因Oct4與Nanog於3T3纖維母細胞與小鼠胚幹細胞中再表現之探討
Reactivation of pluripotency-related genes, Oct4 and Nanog, in 3T3 fibroblasts and mouse embryonic stem cells
作者: 張為芳
Chang, Wei-Fang
關鍵字: Oct4;Oct4;Nanog;Embryonic stem cells;Nanog;胚幹細胞
出版社: 畜產學系所
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特異表現於早期胚、胚幹細胞(embryonic stem cells, ES cells)與其他具分化多能性細胞之Oct4與Nanog,已被證實為維持細胞多能性之重要轉錄因子。Oct4上游調控區域序列會因胚發育階段與細胞分化狀態,而有不同之轉錄活性。研究報告指出,分化細胞中並無Oct4與Nanog之表現,但於ES cells中過量表現Oct4與Nanog,亦會促成ES cells之分化。因此,本研究之目的為選殖小鼠Oct4上游調控區,以期為未來基因轉殖胚早期報導基因(reporter gene)之調控序列,並希望藉由Oct4與Nanog表現載體(pCMV-Oct4與pCMV-Nanog),使ES cells過度表現此兩種轉錄因子,以探討它們對胚層分化之調控。試驗一,自小鼠基因組DNA選殖小鼠Oct4啟動子以及Oct4增強子,構築於pCX-EGFP中,藉由綠螢光蛋白(enhanced green fluorescent protein, EGFP)表現分析Oct4上游調控區於不同細胞與早期鼠胚中之轉錄活性。結果顯示,轉染Oct4上游調控區域至ES cells中,其轉錄活性大於轉染至胚胎成纖維母細胞(mouse embryonic fibroblast, MEF)與結腸癌細胞(CT-26)者,且增強子序列能於ES cells中增強Oct4啟動子之轉錄活性。將含Oct4增強子之調控區基因片段,以顯微注射方式注射入至原核期鼠胚,培養至囊胚後觀察到EGFP集中表現於內細胞群,證實此調控區能專一表現於多能性細胞。試驗二,將pCMV-Oct4轉染至小鼠ES cells與3T3胎源性纖維母細胞後,以RT-PCR、西方吸漬法與免疫螢光染色法分析Oct4之表現。結果顯示,轉染pCMV-Oct4之3T3胎源性纖維母細胞可偵測到Oct4之表現,而轉染之ES cells,Oct4之下游基因,如Esrrb, Rif1 與REST 之表現量,亦隨著Oct4表現上升亦有增加之趨勢,但並未發現各胚層特異性基因於轉染之ES cells中表現。試驗三,以與試驗二相同構築之載體表現Nanog,經轉染後發現Nanog能再表現於3T3 cells,且於ES cells中,其下游基因表現亦類似於試驗二。因此,本試驗所選殖之具胚發育階段與細胞分化特異性之基因調控區,以及所構築之表現載體均具有功能性,可於未來作為基因轉殖,以及研究Oct4與Nanog調控細胞多能性試驗之工具。

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.
其他識別: U0005-2208200600054600
Appears in Collections:動物科學系

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