Site-directed mutagenesis of AP1/NFE2 binding sites in HS-26. (A) AP1/NFE2 binding sites (stars) are replaced with a neo gene, which is flanked by LoxP sites (triangles), through homologous recombination (HR). This is the floxed mutation. The selectable marker is removed by Cre-mediated recombination (Cre). This is the ΔNFE2 mutation. Solid boxes represent exons of the gene C16orf35. The restriction enzyme sites are StuI (S), HindIII (H), EcoRI (E), XbaI (X), Pst I (P), EcoRV (V), and Bgl I (B). (B) Murine α-globin locus with the sequence of the ΔNFE2 mutation. (C) Southern blot analysis of tail DNA from F2 mice. With HindIII, an external probe (▦) hybridizes to a 4.9-kb wild-type band (WT) or a 6.7-kb band (floxed). With XbaI, an internal probe (▨) hybridizes to a 1.8-kb band (WT) or a 2.4-kb band (ΔNFE2). The probes are shown in panel A.