Wild-type Dot1l rescues MLL-AF9 colony formation ability, but not methyltransferase inactive Dot1l (RCR). (A) Genotyping of transduced bone marrow cells after the second round. PCR reaction showed high excision efficiency of endogenous Dot1l with 4-OHT treatment in all cells. (B) Quantitative PCR of exogenous Dot1l expression. All constructs showed expression compared with Neo vector alone. Values were normalized to 5S rRNA internal control. Data are mean plus or minus SD. (C) Western blot of H3K79me2 after second round. Western blot showed restoration of H3K79me2 with the introduction of exogenous wild-type Dot1l but not with RCR. Histone 3 blot was used as loading control. (D) Colony formation on methocult plates. 4-OHT-treated Dot1l^F/F MLL-AF9 cells with wild-type Dot1l were able to form colonies, but not those with RCR. (E) Bar graph of colony count at final round. Data are mean ± SD.