Figure 6

Methylation analysis of all-in-one Tet-ON LVs in hESCs.

DNA from CESTnl2- (a) and CEETnl2- (b) transduced hESCs were extracted at day 15 post-transduction and converted with sodium bisulfate (see M&M for details). Plots showing Dox responsiveness of CESTnl2 and CEETnl2 at the time of lysis are shown at the top of (a,b) respectively. The converted DNA was subjected to PCR using the primers pair indicated in each figure and sequenced. The drawing below the LV schemes (bottom panels in (a,b)) represents the CpG islands contained in the promoter region that was analyzed. Black and white boxes represent methylated and unmethylated CpG respectively. It can be observed that SFFV is highly methylated in AND-1 (a) while hEF1α is highly resistant to methylation (b). (c) TetR expression is restricted to the CEET-transuced hESCs. hESCs (AND-1) transduced with the CESTnl2 and CEETnl2 were lysed and analyzed for TetR expression by Western Blot (see M&M for details).