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 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.
Highlights
The population in a culture of pluripotent cells is not homogenous; an appropriate reporter system, which can screen out the pluripotent stem cell (PSCs) from other metastable stem cells or even completely differentiated somatic cells, is necessary
The distribution pattern of CpGs appeared unique in the porcine Oct4 upstream regulatory sequences (URSs), and three CpG islands were only predicted around pig Oct4 proximal promoter (PP) (Figure 1(b))
The distal enhancer (DE) was located within the CR4 region and the proximal enhancer (PE) was located within the CR2 region (Supplementary Figure 1)
Summary
The population in a culture of pluripotent cells is not homogenous; an appropriate reporter system, which can screen out the pluripotent stem cell (PSCs) from other metastable stem cells or even completely differentiated somatic cells, is necessary. By monitoring the fluorescence signal, the expression of pluripotency-related genes could be determined and the pluripotent cells could be isolated from the heterogenous cell population without additional staining processes [4]. Upon differentiation of PSCs, Oct expression was gradually reduced and silenced along with epigenetic modifications [6]. The silenced Oct in differentiated somatic cells can be reactivated by several reprogramming processes such as fusion-induced reprogramming, somatic cell nuclear transfer (SCNT), or generation of induced pluripotent stem cells (iPSCs) [7, 8], suggesting the importance of Oct in maintenance and
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