Generation of EZH2Y641F transgenic mice. (A) Schematic representation of the construct used to generate the mice. (B) Southern blot using a probe specific for the human growth hormone (hGH) sequence in the transgene performed on 3 transgenic founder mice (1-3), 1 nontransgenic littermate (4), C57Bl6 control (WT) DNA, and C57Bl6 control DNA spiked with 10 pg of plasmid DNA containing the transgenic construct. Genomic DNA was digested using BamHI. Founder 1 carries multiple integrations of the transgenic construct at 2 integration sites; founder 2 contains 1 copy of the transgenic construct in 1 integration site; and founder 3 has 2 copies of the transgenic construct at 1 integration site. (C) Detection of expression of the transgenic construct by quantitative PCR. The lower panel shows a PCR specific for the transgenic construct, and the upper panel a PCR that detects endogenous Ezh2. (D) Steady-state H3K27me3 and H3K27me2 levels in splenic B cells and sorted B220+IgM+ cells. Nuclear lysates from spleen cells of a mouse transgenic for EZH2Y641F or a WT mouse were probed with an antibody against H3K27me3. Levels of H3 were used as a loading control for histones.
