FIG. 5.

Construction and Characterization of Oct4 GFP reporter ESCs. (A, left) Schema of the Oct4 promoter reporter construct. (A, right) Plasmid map for the N1 Oct4 GFP reporter. (B) (1 and 2) ESCs in suspension culture after transfection with the N1 Oct4 GFP reporter vector and G418 selection. Phase contrast (1) and fluorescence images (2) of the same field showing the high degree of GFP fluorescence in undifferentiated ESCs. (3 and 4) In contrast, when the same ESCs are differentiated for 4-6 days by removal of the 2i inhibitors and LIF, the cell morphology indicates that the ESCs differentiated and the GFP fluorescence decreases. Scale bar 100 μm. (C) qRT-PCR analysis of the Oct4 and EGFP transgene expression. Oct4 expression in undifferentiated cells (red bars) and differentiated cells (blue bars). Undifferentiated pCX-EGFP transgenic ESCs (green bars) were used as a positive control for both Oct4 and EGFP expression. Undifferentiated nontransgenic ESCs (pink bars) were used as a positive control for Pou5F1 expression and as a negative control for EGFP expression. The control, housekeeping gene was PBGD. (D) Immunohistochemistry of N1-Oct4-EGFP reporter ESC line in culture. Top, undifferentiated N1-Oct4-GFP reporter ESCs were stained with an antibody to Oct4 (red) to observe the colocalization of eGFP (green) and Oct4 expression. Bottom panel shows the same ESC line after differentiation induced by withdrawal of PD0325901 (MEK inhibitor), CHIR99021 (GSK3β inhibitor) and leukemia inhibitory factor (LIF) for 6 days. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to counterstain the DNA. The field indicated in the inset is shown at higher magnification on the right. Note that the Oct4 staining is localized to the nucleus, and in contrast the GFP signal is found throughout the cytoplasm (arrows indicates one cell that shows nuclear localization of Oct4 and cytoplasmic staining of GFP). Bottom row: Once the N1-Oct4-EGFP reporter line is differentiated by removal of 2i inhibitors and LIF, the cells change morphology (indicating their differentiation). In addition, the DNA changes to a more heterochromatin state (DAPI staining) and the Oct4 staining is lost. Note that when Oct4 immunofluorescence is decreased, so is GFP fluorescence. Note that some cells continue to express Oct4 and they continue to express GFP (examples indicated by arrows). These data suggest that N1-Oct4-EGFP reporter cells faithfully report Oct4 promoter activity and Oct4 protein expression. Scale bar 100 μm. Color images available online at www.liebertonline.com/scd