Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation
Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the...
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| Format: | Article |
| Language: | English |
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Wiley
2016-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.1155/2016/1390284 |
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| author | Wu-Sheng Sun Ju-Lan Chun Jeong-Tae Do Dong-Hwan Kim Jin-Seop Ahn Min-Kyu Kim In-Sul Hwang Dae-Jin Kwon Seong-Soo Hwang Jeong-Woong Lee |
| author_facet | Wu-Sheng Sun Ju-Lan Chun Jeong-Tae Do Dong-Hwan Kim Jin-Seop Ahn Min-Kyu Kim In-Sul Hwang Dae-Jin Kwon Seong-Soo Hwang Jeong-Woong Lee |
| author_sort | Wu-Sheng Sun |
| collection | DOAJ |
| description | Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population. |
| format | Article |
| id | doaj-art-172f8da5106f44de916c87e6ab4f5d7a |
| institution | OA Journals |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Stem Cells International |
| spelling | doaj-art-172f8da5106f44de916c87e6ab4f5d7a2025-08-20T02:04:52ZengWileyStem Cells International1687-966X1687-96782016-01-01201610.1155/2016/13902841390284Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell DifferentiationWu-Sheng Sun0Ju-Lan Chun1Jeong-Tae Do2Dong-Hwan Kim3Jin-Seop Ahn4Min-Kyu Kim5In-Sul Hwang6Dae-Jin Kwon7Seong-Soo Hwang8Jeong-Woong Lee9Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of KoreaDepartment of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of KoreaDepartment of Stem Cell and Regenerative Biology, College of Animal Bioscience and Technology, Konkuk University, Seoul, Republic of KoreaBiotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of KoreaBiotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of KoreaDepartment of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of KoreaAnimal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of KoreaAnimal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of KoreaAnimal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of KoreaBiotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of KoreaOct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.http://dx.doi.org/10.1155/2016/1390284 |
| spellingShingle | Wu-Sheng Sun Ju-Lan Chun Jeong-Tae Do Dong-Hwan Kim Jin-Seop Ahn Min-Kyu Kim In-Sul Hwang Dae-Jin Kwon Seong-Soo Hwang Jeong-Woong Lee Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation Stem Cells International |
| title | Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation |
| title_full | Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation |
| title_fullStr | Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation |
| title_full_unstemmed | Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation |
| title_short | Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation |
| title_sort | construction of a dual fluorescence reporter system to monitor the dynamic progression of pluripotent cell differentiation |
| url | http://dx.doi.org/10.1155/2016/1390284 |
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