Generation of mice with mutant Dot1L alleles. (A) Schematic representation of the gene trap construct used to generate the mutant Dot1L alleles (KO). The numbered boxes represent exons of the Dot1L gene. Arrows represent PCR primers used to amplify DNA from ES cells to identify the exact point of insertion of the gene trap. (B) PCR genotyping with the use of DNA from embryos generated by interbreeding heterozygous Dot1L mutant mice. PCR primers were generated that distinguished WT and KO alleles. As expected, both alleles were detected in heterozygous embryos (lane 3). (C) Absence of H3K79 methylation in MEFs from Dot1L^−/− embryos. MEFs were derived from E10.5 embryos and either serum-starved for 24 hours (left bands) or treated overnight with 20% FCS (right bands). Histones were acid-extracted from cells, and proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Blots were probed with antibodies against histone H3 lysine 79 mono- (H3K79me), di- (H3K79me2), and tri-methylation (H3K79me3). Other antibodies against histone H3 modifications were used to probe the blot: unmodified histone H3 (H3), dimethylated lysine 4 (H3K4me2), dimethylated lysine 9 (H3K9me2), dimethylated lysine 27 (H3K27me2), and lysine 36 (H3K36me2).