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. Author manuscript; available in PMC: 2013 Jan 7.
Published in final edited form as: Cell Stem Cell. 2008 Feb 14;2(3):230–240. doi: 10.1016/j.stem.2008.02.001

Figure 1. Development of a doxycycline-inducible lentiviral system and assessment of the temporal requirement for reprogramming factors.

Figure 1

(A) Schematic drawing of the inducible lentivirus containing a doxycycline-controllable promoter (tetOP-CMV) and a unique EcoRI restriction site to insert cDNAs. (B) to (E) Morphology of infected Oct4-GFP tail-tip fibroblasts cultured with doxycycline for 0, 3, 6 and 9 days. Arrow in (C) indicates a microcolony. (F) GFP fluorescence image of colonies shown in (E). Note weakly GFP+ colony (outlined in green) with smooth borders next to a GFP− colony with differentiated appearance (outlined in red). (G) 3-week-old coat color chimera generated from blastocyst injection of iPS cells made with inducible lentiviruses shown left to a non-chimeric littermate. (H) Experimental outline to determine temporal requirement of reprogramming factors. Fibroblasts heterozygous for ROSA26-rtTA and Oct4-GFP alleles were infected with the four lentiviruses (LV), doxycycline (dox) was added for a period of 0–13 days and GFP+ iPS colonies were scored on day 20. (I) Effect of duration of doxycycline expression on the number of GFP+ (green bars) and GFP− (red bars) colonies present at day 20. (J–M) Brightfield and fluorescence images of a representative GFP+ iPS colony (J, K) as well as a GFP− differentiated colony (L, M) at day 20.