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

Figure 3

Establishment of transgenic cell lines stably expressing EGFP and mCherry (RFP). (a) A schematic illustration of the constructed ΔDE-pOG2-ΔPE-pOm2 vector using the porcine Oct4 promoter. DE: distal enhancer; PE: proximal enhancer. (b) Genotyping the Oct4 promoter-driven transgenes from genomic DNA. PC: positive control; DW: distilled water, negative control; WT: wild type; TG: transgenic cell lines. Lanes 3–6, fluorescence-positive cell lines established from wild type F9 cells, P19 cells, mESCs, and MEFs. (c) Reverse transcriptional PCR analysis of pluripotency markers in fluorescence-positive cell lines. (d) Surface alkaline phosphatase activity in transgenic F9 cells, P19 cells, and mESCs was measured using BCIP/NBT. (e) Typical pluripotency markers, Nanog and Oct4, were assayed by immunofluorescent cytochemical staining in transgenic F9 cells, P19 cells, and mESCs. Scale bars indicate 20 μm.

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