Fig. 6. Myc-maintained ES cells retain pluripotency. (A) Myc^T58AER R1 EGFP ES cells were maintained in the presence of 4OHT for 30 days, in the absence of LIF/Wnt3a CM and then grown as EB suspensions for up to 8 days by withdrawing 4OHT in the absence (top panel) or presence (bottom panel) of LIF. Cell lysates were subject to immunoblot analysis, probing with anti Myc, Oct4 and ß-tubulin antibodies. (B) Myc^T58AER ES cells grown in the presence of 4OHT for 30 days (as in A) were stained for AP activity or grown for an additional 7 days in the absence of 4OHT. (C) GFP marked R1 myc^T58AER ES cells grown for 30 days in the presence of 4OHT (-LIF; as in A) then used for injection into blastocyst stage C57BL/6 embryos that were transferred into females and allowed to develop to 12.5 dpc, at which time they were analysed. Chimeric (bottom) and non-chimeric (top) embryos where EGFP R1 myc^T58AER maintained ES cells had widely integrated into the three germ layers. (D) Dark field view of the same embryos. (E) Tissues were dissected from EGFP-positive embryos and analyzed at higher magnification. (F) The ability of myc^T58AER ES cells to generate liveborn chimeras was confirmed by analysis of coat color. Host C57BL/6 embryos give rise to black coat color, whereas sv129 (R1 ES line) contribute to a sandy-colored coat. Chimeras shown were generated by contribution of LIF maintained (left) and 4OHT maintained (right) myc^T58AER cells.