Figure 1.

Generation of self-renewing iPSCs expressing low levels of Sox2

(A) Diagram of experimental design. rMKO and rMKOS indicate use of retroviral (r) vectors containing reprogramming cMyc (M), Klf4 (K), Oct4 (O), and Sox2 (S) transgenes. + EV (empty vector) or + Sox2 represent use of a PiggyBac plasmid containing a CAG promoter driving constitutive expression of either an empty or Sox2 transgene, respectively.

(B) Phase and GFP images of emerging Rex1-GFP + iPSC colonies (n = 3).

(C) Rex1-GFP + colony counts for indicated genotypes (n = 3).

(D) Phase and GFP images of Sox2^−/− rMKOS iPSCs in 2iLIF post-selection. Rex1-GFP ESCs are shown as control.

(E) qRT-PCR analysis for indicated pluripotency associated factors in iPSC and control ESC lines.

(F) Western blot of Sox2 (≈40kDa) and Tubulin (≈50kDa) in iPSC and control ESC lines in 2iLIF with Sox2 quantification relative to ESCs, normalized to tubulin. Gap in Western blot represents removal of a non-relevant lane and image corresponds to same film exposure.

(G) qRT-PCR of retroviral Sox2, Oct4, Klf4, and cMyc expression in Sox2^−/− rMKOS iPSCs relative to preiPSCs.

Scale bars = 200μm.

Error bars indicate standard deviation of replicate qPCR reactions (n = 3).