Analysis of the variant ANK-1 promoters in transgenic mice. Each promoter was linked to a human Aγ-globin reporter gene and injected into fertilized mouse eggs. Founder animals were bred to generate F1 animals for analysis of the copy number and γ-globin mRNA level. (A) DNA, Southern blot analysis of wild type (+C) and low-copy-number animals with the variant ANK-1 promoters (GC, GCGGGTGAG; GG, GGCGGTGAG; 3G, GGGGGTGAG). The estimated copy number is indicated above the image of the Southern blot. RNA, RNase protection analysis of γ-globin mRNA levels in wild type (+C) and low-copy-number animals with the variant ANK-1 promoters (GC, GG, and GGG). (B) The relative levels of human γ-globin and endogenous mouse α-globin mRNA per transgene copy. Wild type (WT): n = 16; GC, n = 5; GG, n = 6; and GGG, n = 5). *, P < 0.01 compared to the wild type. (C) Fluorescence activated cell sorting analysis of human γ-globin protein in erythrocytes from wild type (+C) and low-copy-number animals with the variant ANK-1 promoters. −C, erythrocytes from a transgene negative littermate control. +C, erythrocytes from a mouse carrying >20 copies of the wild-type ANK-1 promoter/γ-globin transgene. GC, GG, and GGG, erythrocytes from mice carrying one to six copies of the indicated variant ANK-1 promoter/γ-globin transgene. Analysis results for all of the animals are presented in Table 2.