Fig. 5. NANOG overexpressing cells adopt markers of primitive ectoderm cells. (A) The ICM (inner cell mass) and epiblast (primitive ectoderm) can be distinguished by the differential expression of a subset of marker genes. ICM has a high expression of Rex1 and Gbx2, and low expression of Fgf5. Epiblast has low expression of Rex1 and Gbx2, and upregulation of Fgf5. (B) Real-time RT-PCR analysis was performed on markers that distinguish between ICM and primitive ectoderm cells. Expression was examined on wild-type cells and on NANOG overexpressing clones (shown as the average of three separate clones). Although GAPDH and OCT4 did not show a significant change, REX1 and GBX2 were significantly downregulated, and FGF5 was significantly upregulated following NANOG overexpression (^*P<0.05). (C) Semi-quantitative RT-PCR analysis was executed on the markers described in B. Expression was examined in wild-type cells, NANOG overexpressing clones and revertant clones from which the transgene was excised. Shown are representative pictures of the examined clones. (D) RT-PCR analysis was performed on Nanog overexpressing MESCs. A comparison was performed between two separate MESC lines (E14Tg2a and RF8) and clones overexpressing Nanog derived from them.