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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Nat Methods. 2011 Nov 13;8(12):1056–1058. doi: 10.1038/nmeth.1775

Figure 1. Generation of inducible cell lines.

Figure 1

(a) The schematic depicts the cloning strategy to generate inducible lines with epitope tagged genes. Coding sequences lacking the STOP codon are V5-tagged at the C-terminus. (b) Overview of embryonic stem cell (ESC) directed differentiation. Differentiating cells become motor neuron progenitors (pMNs) at Day 4 and produce motor neurons (MNs) from Day 5–7. Doxycyline (Dox) is added late on Day 3 or Day 4 to mimic the expression pattern of the endogenous Olig2 and Hoxc9 respectively. Olig2-V5 is analyzed at Day 4 and Hoxc9-V5 or FlagB-Hoxc9 at Day 5. (c) Nkx2.2 staining in control or Olig2-V5 expressing cells at Day 4 of differentiation under high Hh concentration (500 nM). (d) Pax6 staining in control or Olig2-V5 expressing cells at Day 4 of differentiation under low Hh (5 nM). (e) iHoxc9-V5 and iFlagB-Hoxc9 day 5 embryoid bodies stained with anti-Hoxc4, V5 and Flag antibodies.