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Figure 1


Fig. 1. Generation of a tetracycline-regulated Nodal-expressing ES cell line. (A) The construct and strategy for the regulated expression of Nodal in ES cells (Tet-off system). (B) Outline of definitive endoderm and mesoderm differentiation using Nodal-expressing ES cells. (C) FACS analysis of cells with GFP expression during stages 1 and 2. (D) Bright-field and GFP immunofluorescent images of cells during stage 2. ES cells were plated on feeder-free, type IV collagen-coated dishes. Most ES cells expressed GFP throughout differentiation. (E) Western blot analysis of Nodal protein expression during stages 1 and 2. Both the mature form (13 kDa) and the precursor of Nodal protein are detected at stage 1 and is diminish at stage 2 in Tet-on/Nodal-off ES cells. (F) Nodal gene expression by real-time RT-PCR analysis during stages 1 and 2. The level of Nodal expression in Nodal-on ES cells is roughly 2000 times higher than that in the Nodal-off control cells. (G) The total cell number relative to the initial number during stages 1 and 2. Nodal expression shows an insignificant effect on cell growth at stage 2.





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