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. 2008 Dec 29;29(6):1635–1648. doi: 10.1128/MCB.01735-08

FIG. 1.

FIG. 1.

Production of PBGDAγ-YAC mice and characterization of transgenic lines. (A) The human β-globin locus in a YAC was modified by replacement of the 887-bp proximal Aγ promoter (pr. Aγ), from positions −838 to +49 (SapI to BsaHI fragment), with the equivalent region (887 bp) of the erythroid-specific PBGD promoter (pr. PBGD) that spans positions −815 to +72 (BamHI fragment). (B) Analysis of the PBGDAγ-YAC construct by use of different restriction enzymes (EcoRI, HindIII, BamHI, PstI, and BstEII) on the modified human β-globin YAC obtained by homologous recombination in yeast. Southern analysis was carried out on DNA isolated from yeast containing the PBGDAγ-YAC mutant (mut) or the wild-type human β-globin YAC (wt). The following five probes were used: (i) HS2 probe (EcoRI; 10.4 kb), (ii) ɛ probe (EcoRI; 3.7 kb) containing pSP73 vector sequences hybridizing to the YAC arm (Ya), (iii) 3′ Gγ probe (BstEII; 3.7 kb for wild type and 3.1 kb for PBGDAγ), (iv) γ probe hybridized to Gγ (EcoRI; 1.6 and 7.0 kb) and to Aγ (EcoRI; 2.6 kb for wild type and 2.1 kb for PBGDAγ), and (v) β probe (SphI fragment) for the β-globin (EcoRI; 5.5 kb and 3.8 kb) and δ-globin (EcoRI; 2.3 kb) genes. Analysis by PFGE was also carried out on KpnI or SfiI/SalI fragments, using probes for the YAC left arm (a), LCR (b), γ (c), and the YAC right arm (d), or on the full-length sequence (∼150 kb), using the γ probes (c). No rearrangements in the β-globin locus were detected from the Aγ promoter substitution homologous recombination steps. (C) Representative transgenic line (PY) analysis of transgene integrity by Southern blotting of EcoRI-digested genomic DNA compared to DNA of the wild-type β-YAC transgenic line (Y), monitored with probes i, ii, and iv (see above) and with a β probe (SphI-BamHI fragment) hybridized to the β-globin gene (5.5 kb) and to the δ-globin gene (2.3 kb). The presence of both the left and right arms of the YAC was detected by a multiplex PCR (204 and 183 bp for the left and right arms, respectively), as shown for both sides of the locus. Seven transgenic lines displayed all expected bands.