shRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. Lentiviral vector LL3.7 containing DNMT1 shRNA and GFP sequences was used to transduce differentiating cells on day +5. On day +7 cells expressing GFP were sorted and allowed to differentiate into erythroid cells. Controls for this experiment were cells from the same initial culture transduced with vectors carrying a mismatched version of the DNMT1 shRNA, an empty vector, or cells that were untreated. Effects of DNMT1 shRNA on (A) DNMT1 steady-state mRNA levels during in vitro differentiation, (B) DNMT1 protein levels as determined by Western blot, (C) γ-globin promoter DNA methylation, (D) LINE element DNA methylation, (E) γ-globin steady-state mRNA, and (F) Hb levels as determined by HPLC at the end of differentiation. CD34⁺ cells from a single healthy donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.