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. Author manuscript; available in PMC: 2014 Apr 15.
Published in final edited form as: Nat Methods. 2009 Dec 13;7(1):53–55. doi: 10.1038/nmeth.1409

Figure 1. An ES-cell based reprogramming system.

Figure 1

(a) Genomic configuration of Collagen-OKSM ESCs with the cDNA of the optimized reverse tetracycline-dependent transactivator (M2-rtTA, protein product represented by dark triangles) targeted to the constitutively active ROSA26 locus and a polycistronic cassette encoding Oct4 (O), Klf4 (K), Sox2 (S) and c-Myc (M) targeted to the Col1a1 locus under control of a tetracycline-dependent minimal promoter (tetOP). In the presence of doxycycline (dox) M2-rtTA binds to tetOP, thereby inducing expression of the four reprogramming factors. (b) Outline for a secondary reprogramming system based on Collagen-OKSM ESCs or Lenti-OKSM iPSCs. Chimeric animals are generated by blastocyst injection and ESC-derived or iPSC-derived somatic cells are isolated and reprogrammed into iPSCs by culture in dox-containing media. (c) Brightfield images of a chimera-derived MEF-iPSC clone before picking (P0, left) and after passaging (P3, right). (d) Alkaline phosphatase staining of colonies derived from Collagen-OKSM MEFs or MEFs carrying two copies of a dox-inducible lentiviral vector encoding the same polycistronic cassette (Lenti-OKSM). (e) Graph showing the efficiency of iPSC formation from Collagen-OKSM MEFs (blue bar) and Lenti-OKSM MEFs (green bar), respectively. (f) Fluorescent images of Collagen-OKSM and Lenti-OKSM MEFs cultured for two days in the presence of doxycycline and stained with an antibody against Oct4 (red) and DAPI (blue). Numbers indicate the percentage of Oct4+ cells. Scale bars = 100 μm. (g) Histological section through a teratoma derived from Collagen-OKSM MEF-iPSCs showing differentiation in ectodermal (EC, keratinized epithelium), endodermal (EN, glandular structures) and mesodermal (MS, muscle) derivatives. (h) Image of an adult coat color chimera (red arrow) derived from MEF-iPSC. Also shown are two non-chimeric BDF1 mice. (i) Image of an E13.5 embryo derived after injection of MEF-iPSCs into tetraploid blastocysts (“all iPSC” fetus). Scale bars, 500 μm (c), 10 mm (d), 100 μm (f), 50 μm (g), 2 mm (i).