Transgene copy number-dependent rescue of murine β-globin knockout mice carrying a 183 kb human β-globin BAC genomic fragment

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human β-globin (huβ-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the huβ-globin locus at a single integration site. Steady state levels of huβ-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background (huβ+/0, muβth-3/+) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the huβ-globin locus, and in one transgenic line, which was hemizygous for six copies of the huβ-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% huβ-globin production from the intact huβ-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific β-thalassemia mutations in the huβ-globin locus.