Fig. 1.

Generation of murine iPS cells using a single 4F2A polycistronic virus. (A) FUW lentivirus constructs tested by transient transfection. In total, four 2A peptides (F2A, T2A, E2A, and P2A) were used. (B) Transient transfection of 293 cells with FUW 2A lentiviruses. Cells were harvested after 48 h and analyzed by Western blot (WB). Efficient protein expression was observed in all constructs tested, indicating four unique 2A peptides support robust protein expression. Note: Sox2 protein is not detected in ES cells because only a short exposure was used. (C) Schematic of the 4F2A DOX-inducible lentivirus containing three types of 2A peptides (P2A, T2A, and E2A). Murine cDNAs for Oct4, Sox2, Klf4, and c-Myc. This particular sequence of factors and 2A peptides is subsequently referred to as “4F2A.” (D) RT-PCR anaylsis of mRNA induction in cells transduced with OSKM 4F2A + rtTA for 3 days. Total Oct4 or Sox2 induction was used to test levels of 4F2A induction relative to ES cells. E2A-cMyc primers were used to detect viral-specific transcripts. Error bars represent SD of the mean of triplicate reactions. (E) Western blot analysis of MEFs transduced with 4F2A + rtTA for 3 days. Cells infected with 4F2A DOX-inducible lentivirus + rtTA produce all four reprogramming factors upon addition of doxycycline (DOX).