Fig. 3. EG cell production from AKT-MER-expressing PGCs in the absence of bFGF and SCF. (A) Increased number of AKT-MER-expressing PGCs in the absence of bFGF. E11.5 gonad cell suspensions were seeded onto Sl/Sl⁴-m220 feeder cells and cultured with LIF but without bFGF. The cells of 0.1 embryos were added to each well. The percentage change in PGC number was calculated as described in Fig. 2C. The 4OHT-treated transgenic PGCs proliferated significantly more than did the untreated transgenic PGCs (mean±s.e.m.; ^*P<0.005, Student's t-test; n=4) and wild-type PGCs cultured with the corresponding concentrations of 4OHT (P<0.005 for 100 nM, 300 nM and 1000 nM). (B) Primary EG cell colony formation by E11.5 PGCs of transgenic mice without bFGF. Significantly more EG colonies were generated in the 4OHT-treated transgenic cultures than in the untreated transgenic PGCs (mean±s.e.m.; ^#P<0.001, ^*P<0.005, ^$P<0.05, Student's t-test; n=4). (C) Alkaline phosphatase-positive primary EG cell colonies generated from transgenic PGCs treated with 300 nM 4OHT in the absence of bFGF. Cell suspensions of transgenic gonads at E11.5 were seeded onto Sl/Sl⁴-m220 feeder cells and cultured with LIF alone for 5 days. Scale bar: 100 µm. (D) Transgenic EG cell lines established without bFGF from the primary PGC cultures in C. The EG cells formed round, multilayered colonies (left) and were EGFP positive (right). Scale bar: 100 µm. (E) EG cell lines established from E11.5 PGCs of transgenic mice without bFGF and SCF. Gonad cell suspensions were seeded onto MEFs, cultured with LIF and 300 nM 4OHT, and then passaged to tertiary cultures. Scale bar: 100 µm. (F) Mixed culture of wild-type and transgenic cells. Gonad cell suspensions of E11.5 wild-type and transgenic mice were cultured on Sl/Sl⁴-m220 feeder cells in the presence of LIF and 300 nM 4OHT, and passaged to tertiary cultures. All of the EG cell colonies were positive for EGFP (right). Scale bar: 250 µm.