Conditional targeting of the mouse Suz12 gene. (A) The WT (+) Suz12 locus, indicating exons 4-7 is shown, with the fl conditional allele, and recombined knockout allele (Δ) following cre-mediated excision. The WT Suz12 locus was modified after the insertion of a targeting vector containing 2 loxP sites flanking exon 5 of the Suz12 gene, which also contained a neomycin cassette flanked by 2 Flip recombinase target sites located after the 3′-end of exon 5 and just upstream of the second loxP site. Expected fragment sizes in Southern blot analysis and PCR-based genotyping are shown. (B) PCR analysis of genomic DNA extracted from BM and spleen of Suz12/CreER^T2 mice 8 days after tamoxifen treatment, using a primer combination that distinguishes WT, fl, and recombined (Δ) alleles. (C) Immunoblot analysis of Suz12 and Ezh2 protein in the thymus, spleen, and BM of Suz12^fl/+/CreER^T2 and Suz12^fl/fl/CreER^T2 mice 8 days after tamoxifen treatment. Actin was used as loading control. (D) Immunoblot analysis of Suz12, Ezh2, and H3K27me3 in cultured fetal liver cells from Suz12^fl/fl/CreER^T2 E14 embryos. Cell lysates were prepared 24 hours after 4-hydroxy tamoxifen (4-OH Tam]) or vehicle treatment. Total histone H3 and actin were used as loading controls.