Fig. 1. Efficient neurogenesis of ES cells under serum-free LIF-deficient conditions. ZHTc6 ES cells were established (Niwa et al., 2000) in which expression constructs for a tetracycline (Tc)-regulated transactivator (tTA) transgene and a tTA-responsive Oct3/4 transgene were introduced. The ZHTc6 cells with no transgene expression in the presence of Tc showed induced Oct3/4 expression the level of which is comparable to that from the endogenous allele. Withdrawal of Tc triggers the transgene expression, which induces Oct3/4 expression at a level 50% above that of Oct3/4 expression in normal ES cells. (A) ZHTc6 cells were cultured in serum-free N2-supplemented DMEM/F-12 without LIF for 8 days on poly-L-ornithine/fibronectin (O/F)-coated dishes in the presence (a) or absence (b) of Tc. Cells were stained with an anti-MAP2 antibody (green) and Hoechst 33258 (blue). Upregulation of Oct3/4 induced by withdrawal of Tc promoted neurogenesis of ES cells. Scale bar, 50 µm. (B) ZHTc6 cells were cultured as in A with or without Tc for 10 days. The frequency of MAP2-positive cells with respect to the total number of cells was analyzed every 2 days. Vertical bars indicate the s.d.