Wild type or Dnmt1-/- ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 days and analyzed for GFP recombination and expression. A. Genomic DNA from the two cell lines was PCR-amplified with non-recombinant (5’-UnRec) and recombinant (5’-Rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. RealTime-PCR of the same samples carried out as described in Materials and Methods. B. Bivariate FACS analysis of cells transfected with I-SceI (GFP vs Side Scatter, SSC). The gating of GFP⁺ cells was created to exclude up the 99.5% of WT, untransfected ES cells. The same gating applied to Dnmt1-/- cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1-/- cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild type ES but did not enhance the expression of GFP in the Dnmt1-/- cells C. histogram showing the fraction of GFP⁺ cells derived from 3 experiments. To get reliable values of differential GFP fluorescence in ES and Dnmt1-/- cells, we compared the percentage of GFP+ cells, normalized for the transfection efficiency in 6 experiments (3 in duplicate) with the Wilcoxon Kruskal-Wallis Test, * p < 0.012 versus wild type.

https://doi.org/10.1371/journal.pgen.1006605.g001