FIGURE 4.

Generation of dFib-OCT4^GFP–ind lines with doxycycline-inducible expression of different subsets of reprogramming factors. A, morphology of the dFib-OCT4^GFP–ind fibroblasts-like cells (at passage 7) after embryoid body-mediated differentiation. Scale bar = 50 μm. B, real-time PCR analysis was performed for the pluripotent markers OCT4, SOX2, NANOG, the reprogramming factors KLF4 and cMYC, the fibroblast marker COL1A1, and for GFP. Data are shown as relative means ± S.D. of two biological replicates analyzed in triplicate. C, dFib-OCT4^GFP–indSKC, dFib-OCT4^GFP–ind-OKC, and dFib-OCT4^GFP–ind-OSK cells were infected with retroviruses encoding OCT4, SOX2, and cMYC, respectively, and either treated or not treated with 100 ng/ml of doxycycline for 48 h. Real-time PCR analyses were performed to detect the transcripts corresponding to the four reprogramming factors. Note that there is no residual expression from any of the factors delivered by retroviruses to generate the original primary hiPSC lines. Data are shown as relative means ± S.D. of two biological replicates analyzed in triplicate.