Figure 2. Characterization of iPS cells generated using a single lentiviral vector.

(A) Representative pictures of TTFs from a Sox2-GFP M2rtTA mouse transduced with the inducible STEMCCA vector, in the absence (left panel) or presence (right panel) of doxycycline induction. Images were acquired on day 14 post-infection. (B) Representative pictures of iPS cells derived using the constitutive pHAGE-EF1α-STEMCCA (EF1α) or the inducible pHAGE-Tet-STEMCCA vector (Tet) show colony morphology (Phase), high alkaline phosphatase activity (Alk Phos), SSEA1 immunostaining, and Sox2-GFP reporter gene expression. (C) Expression of ESC ‘marker’ genes detected by RT-PCR in four representative iPS cell clones generated by the constitutive (EF1α) or inducible (Tet) STEMCCA vector. Nat1 is a constitutively expressed gene and serves as a control for loading. Representative samples from unmanipulated tail tip fibroblasts (TTF) and mouse ES cells are also shown. An iPS cell sample prepared without RT was used as negative control (-RT). (D) Southern blot analysis of genomic DNA (gDNA) purified from 6 representative iPS clones produced with the constitutive (EF1α) or inducible (Tet) vector. gDNA was digested with BglII to obtain a band of 6.7Kb in the EF1α colonies or 8.3Kb in the Tet colonies, representing most of the proviral genome. For control, pHAGE-Tet-STEMCCA plasmid DNA representing 1 or 2.5 copies of the insert was digested with BglII. A single band of the expected size of the proviral gene insertion is present in all clones. The density of each band indicates between 1–3 proviral integrations in each clone.