Cellular Reprogramming Employing Recombinant Sox2 Protein
Figure 4
Cellular and molecular characterization of iPS clones derived by Sox2 protein transduction into OKC-MEFs. (a) Pictures of isolated cell lines Sox2-piPS-1 (upper row) and Sox2-piPS-2 (lower row) exhibiting brightfield (BF), staining against pluripotency-associated marker SSEA-1 and native GFP fluorescence. Sox2-piPS-1 cell line was clonally isolated from 400 nM Sox2-TAT treatment from day 1 to 5, and Sox2-piPS-2 was derived from 200 nM condition (day 5 to 10) Scale bar = 100 μm. (b) PCR analysis of genomic DNA demonstrating genomic integration of Oct4 and Klf4 transgenes. As expected, no transgenic Sox2 was detected in Sox2-piPS clones excluding possibility of contamination. (c) RT-PCR analysis demonstrating transgene silencing in Sox2-piPS cells. Primers specific for transgenic Oct4, Sox2, Klf4, and c-Myc were used. Additionally, we analyzed endogenous Oct4, Sox2, and Nanog transcripts. RNA preparations from infected (Inf.) and uninfected MEFs as well as ES cells served as controls.
