title: Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation.
abstract: 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 mouseOct4gene, which were demonstrated to controlOct4expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pigOct4and 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.
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea; Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Wu-Sheng
authors.initials: WS
authors.lastname: Sun
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Ju-Lan
authors.initials: JL
authors.lastname: Chun
authors.affiliation: Department of Stem Cell and Regenerative Biology, College of Animal Bioscience and Technology, Konkuk University, Seoul, Republic of Korea.
authors.forename: Jeong-Tae
authors.initials: JT
authors.lastname: Do
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
authors.forename: Dong-Hwan
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
authors.forename: Jin-Seop
authors.initials: JS
authors.lastname: Ahn
authors.affiliation: Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of Korea.
authors.forename: In-Sul
authors.identifier: 0000-0003-2949-3075
authors.initials: IS
authors.lastname: Hwang
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of Korea.
authors.forename: Dae-Jin
authors.identifier: 0000-0002-9512-2283
authors.initials: DJ
authors.lastname: Kwon
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, Wanju 565-851, Republic of Korea.
authors.forename: Seong-Soo
authors.identifier: 0000-0002-0512-7049
authors.initials: SS
authors.lastname: Hwang
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
authors.forename: Jeong-Woong
authors.identifier: 0000-0002-2649-9677
authors.initials: JW
authors.lastname: Lee