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Stem Cells International
Volume 2016 (2016), Article ID 1390284, 11 pages
Research Article

Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation

1Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
2Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of Korea
3Department of Stem Cell and Regenerative Biology, College of Animal Bioscience and Technology, Konkuk University, Seoul, Republic of Korea
4Animal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of Korea

Received 17 August 2016; Revised 6 October 2016; Accepted 18 October 2016

Academic Editor: Gary E. Lyons

Copyright © 2016 Wu-Sheng Sun et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.