Expression of human β-globin genes in knock-in mice.

(A) Total RNA was prepared from spleens of 1-month-old anemic mice (N = 4 for each genotype). Expression levels of the human β(γ)- or β(ε)-globin genes (analyzed by common primer set targeted at β-globin sequence; BT-4S1 and BT-4A1) and endogenous mouse α (mα)-globin gene were analyzed by qRT-PCR. The ratio of hβ/mα-globin genes was calculated (the expression value of the β(γ)-globin was set at 1). P values (vs β(γ): *(B) Total RNA was prepared from spleens, livers and kidneys of 1-month-old anemic mice (N = 4 for each tissues). Expression of mα-globin, β(γ)-globin and endogenous mouse (m)GAPDH genes was analyzed by qRT-PCR. The expression levels of mα- (left panel) or β(γ)-globin (right) genes, both compared to that of mGAPDH gene were calculated (the expression values in spleen samples were set at 100). (C and D) ChIP was conducted for CTCF in the spleen cells of anemic animals bearing both LCR+β(ε) and β(γ)+3’HS1 alleles. The Necdin gene and the H19 ICR sequences were analyzed as negative and positive controls, respectively. Quantitative PCR was repeated at least three times for each sample. Fold enrichment of CTCF relative to IgG control (average values with S.D.) was calculated and graphically depicted (average value of negative controls was set at 1.0). P values (vs Necdin): *<0.05; **<0.01.