Analysis of γ-globin expression at E14.5 and E18.5. (A) S1 nuclease protection assay on E14.5 and E18.5 fetal liver RNA, detecting human γ-globin (h-γ) and human β-globin (hβ) messenger RNA (mRNA). Mouse α-globin was used as a loading control. (B) QRT-PCR analysis of human globin expression. γ/γ+β ratios are significantly higher in all mutant mice compared with the controls (n = 3 to 5 for each genotype and each time point). (C) Western blot on E18.5 blood samples detecting expression of γ-globin protein. Actin was used as a loading control. (D) Immunohistochemistry of γ-globin expression (brown) in E18.5 blood. The white circles in (A) denote embryos heterozygous for the human β-globin locus transgene. *P < .05 between mutant and control groups; P < .05 between mutant groups is indicated by a color-matched asterisk.