Figure 1. Reprogramming somatic cells from transgenic Col1a1 4F2A mice.

(a) A targeting vector to the 3’ UTR was utilized to deliver four murine reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) as a single dox-inducible polycistronic transgene ¹¹,¹². Red triangles indicate loxP sites. pA indicates poly-adenylation sequence. TetO is tetracycline operator minimal promoter. (b) Southern analysis of DNA obtained from Neo-resistant V19 ES cell colonies was digested with XbaI and probed for correct targeting of the Col1a1 3’ UTR using a 5’ external Col1a1 probe to the genomic sequence outside the targeting vector homology arms ¹¹. Lines #2 and #11 were correctly targeted as indicated by hybridization to 5.3 kb band (untargeted allele ~ 4.7 kb). (c) Col1a1 4F2A iPS cells express pluripotency markers. Bright field (BF) images and immunostaining for pluripotency markers alkaline phosphatase (AP), SSEA1, and Nanog. Scale bar, 1mm. (d) Oct4 promoter using bisulfite sequencing. Open circles indicate unmethylated and closed circles indicate methylated CpG dinucleotides. Shown are representative sequence analyses from Col1a1 4F2A transgenic MEFs, embryonic stem cells (V6.5), and two Col1a1 4F2A iPSC lines: pro-B-derived #1 and liver-derived line #5B. (e) Hematoxylin and eosin staining of teratomas induced after subcutaneous injection of Col1a1 4F2A Liv iPSC #5B and Ker iPSC #2 into SCID mice indicates Col1a1 4F2A iPSCs contribute to all three germ layers. Scale bar, 100μm (f) Postnatal chimeric mice detected by agouti coat color from Col1a14F2A MEF iPSC #11 and MAC iPSC #5.