H2afy gene-targeting strategy and genotyping of cells and mice. (a) Schematic diagrams of the H2afy locus, the targeting vector and the mutant H2afy alleles are shown. The exons are numbered. The targeting construct containing a neomycin (Neo) cassette flanked by two loxP sites (triangles) was inserted in place of exon 2, resulting in the deletion of the first coding exon, which encodes amino acids 1 to 57. The targeted locus lacks the translation initiation codon and half of the sequence coding for the histone region, and should therefore function as a null allele. The first 18 nucleotides (non-coding) of exon 2 were not modified by the targeting in order to maintain the correct splicing of the first exon to the second. This mutation affects both splicing isoforms macroH2A1.1 and macroH2A1.2, because they are generated by alternative splicing of exon 6. Probes used for the Southern blot studies and relevant restriction fragments predicted by digestion of the recombinant are shown. The sizes of the diagnostic fragments for the wild-type (WT) and H2afy mutant alleles are indicated. (b) Southern blot analysis of recombinant embryonic stem cells (digested with Eco RI). The blots were hybridized with the probes shown in (a). (c) Immunodetection of macroH2A1 and macroH2A2 in H2afy-/- and wild-type mice. Nuclear extracts were prepared from various tissues. The protein macroH2A1 was undetectable in the mutant tissue, proving that the targeted mice were null mutants. There was no overexpression of macroH2A2, which was detected only in brain and embryo (data not shown). H2B shows equal protein loading between all lanes. The additional fast migrating bands seen in testis are probably caused by the crossreactivity of the anti-H2B antibody with the testis-specific variant of histone H2B.