Creation of conditional PHD2 allele. (A) Targeting strategy for mouse PHD2. NEO cassette flanked by 2 Frt sites (▵) is excised by fliplase to create floxed allele. Exons 2 and 3 flanked by 2 LoxP sites (▴) are excised by Cre recombinase to create a null allele. DT-A indicates diphtheria toxin-A used to select against nonhomologous recombinants; ■, exon; and □, untranslated region (UTR). Selected restriction sites are shown: EcoRI (RI), HindIII (H), BamHI (B), SpeI (Sp), and NotI (N). One-sided arrows = PCR primers used for genotyping. 2-sided arrows indicate BamHI fragment detected by Southern Blot with indicated probe. (B) Southern blot analysis of tail genomic DNA, digested with BamHI, from mice with indicated genotypes using probe shown in panel A. In the bottom panel, mice contained EIIA-Cre transgene where indicated. (C,D) RT-PCR (C) and immunoblot analysis (D) of MEFs with indicated genotypes. *The faint band is probably a background band, as PHD2 mRNA is undetectable in these cells. (E) Immunoblot analysis of PHD2 flox/flox (F/F);Cre-ER MEFs after treatment with 4-hydroxy tamoxifen (4-OHT; 200 nM) for the indicated time period. (F) Immunoblot analysis of kidney and liver from PHD2 flox/flox;Cre-ER mice after tamoxifen treatment.