Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts

Figure 3

OSK + pTAT-mcMyc treated MEF display the hallmarks of pluripotency. (A) Schedule for pTAT-mcMyc treatment of somatic cells. MEF were infected for 24 hours (“I”) before retrovirus was washed from cells (“W”) and pTAT-mcMyc and control recombinant protein applied (day 0; “P”). Protein was applied daily for 12 days. (B) Oct4-GFP⁺ clone Tatc1 from Oct4/Sox2/Klf4 + pTAT-mcMycs treated cells. (C) Confirmation of alkaline phosphatase expression in clone Tatc1. (D) RT-PCR confirms Tatc1 expresses endogenous markers Oct4, Sox2, cMyc, Klf4, Rex-1, and Nanog (“Endo”). We further confirmed this clone was not contaminated with mcMyc transgene by PCR using genomic DNA template (“T’gene”; all oligonucleotides outlined elsewhere) [1]. (E) Following injection into SCID mice, Tatc1 form teratomas with differentiated cells characteristic of the three germ layers (cartilage characteristic of endoderm, glandular tissue reminiscent of mesoderm, and neural rosettes characteristic of ectoderm). (F) Immunocytochemistry confirms Tatc1 expresses pluripotency marker stage-specific embryonic antigen-1 (SSEA1). (G) Reprogramming of MEF to Tatc1 maintained normal karyotype (40XY). (H) Following aggregation with 2.5 dpc embryos, Oct-GFP⁺ Tatc1 cells contribute to the inner cell mass (labeled “ICM”) of subsequent 4.5 dpc blastocysts (arrow).