Figure 1.

Deletion and Southern blot strategies to generate ΔEyΔβh1 promoter deletion mice. (A, I) Schematic map of the Hbb(d) allele. (arrows) LCR hypersensitive sites. (■) β-like globin genes. (II) Ey and βh1 genes with exons marked. Deleted regions from each gene are indicated by solid lines under the gene names. (III) Ey promoter was replaced by PGK-hygro, and βh1 promoter was replaced by PGK-neo. (▴) Selectable markers were each flanked by 2 convergent lox P sites and were removed by Cre recombinase-mediated deletion. (arrows) Selectable marker transcriptional start and orientation (IV). Cre recombinase also mediated an additional inversion of DNA between the 2 LoxP sites left from the deletion of the selectable markers and resulted in 2 double-promoter deletion alleles, ΔEyΔβh1 and ΔEy(inv)Δβh1. (arrows) Transcriptional orientation of βh0 on each allele. (B) Southern blot strategies and representative Southern blot used to screen for ΔEyhygroΔβh1neo ES clones modified in cis after homologous recombination. Restriction sites for SpeI are marked as S. The probe is denoted as a short line under each map. Note that the expected band for wild-type Hbb(d) measures 42.2 kb, which is not shown on the gel. Each lane represents a single ES clone. (C) Southern blot strategies and representative Southern blot to screen for ΔEyΔβh1 and ΔEy(inv)Δβh1 promoter deletion alleles in mice after Cre-mediated selectable marker deletion in vivo. Restriction sites for NsiI are marked as N, and the probe used is a line underneath each allele. Expected band sizes for each genotype are marked accordingly.