Fig. 6. Examination of mouse ES cells for differentiation ability of Rex1^-/Oct3/4⁺ cells selected with thymidine kinase gene/gancyclovir system into primitive endoderm lineage. (A) Thymidine kinase gene knock-in ES cell lines under selection for puromycin and gancyclovir (GANC). (Left) Cells selected with puromycin (selection for Oct3/4⁺ cells); (right) cells selected with both puromycin and GANC (selection for Oct3/4⁺ and exclusion of Rex1⁺ cells). (B) Expression of marker genes was examined by RT-PCR in puromycin and GANC/puromycin double-selected fractions. (C) Expression of primitive endoderm marker genes in differentiated Rex1⁺, Rex1^- and `reverted' Rex1⁺ cells by withdrawal of LIF. Before starting induction of differentiation, Rex1⁺ cells were selected with blasticidin S whereas Rex1^- cells were selected with GANC and puromycin for 1 week. Reverted Rex1⁺ cells were derived from Rex1^- cells re-seeded and cultured without GANC for 3 days, and then cultured with blasticidin S for 4 days. Scale bar: 100 µm.