Animal models of β-hemoglobinopathies: utility and limitations

Abstract

The structural and functional conservation of hemoglobin throughout mammals has made the laboratory mouse an exceptionally useful organism in which to study both the protein and the individual globin genes. Early researchers looked to the globin genes as an excellent model in which to examine gene regulation - bountifully expressed and displaying a remarkably consistent pattern of developmental activation and silencing. In parallel with the growth of research into expression of the globin genes, mutations within the β-globin gene were identified as the cause of the β-hemoglobinopathies such as sickle cell disease and β-thalassemia. These lines of enquiry stimulated the development of transgenic mouse models, first carrying individual human globin genes and then substantial human genomic fragments incorporating the multigenic human β-globin locus and regulatory elements. Finally, mice were devised carrying mutant human β-globin loci on genetic backgrounds deficient in the native mouse globins, resulting in phenotypes of sickle cell disease or β-thalassemia. These years of work have generated a group of model animals that display many features of the β-hemoglobinopathies and provided enormous insight into the mechanisms of gene regulation. Substantive differences in the expression of human and mouse globins during development have also come to light, revealing the limitations of the mouse model, but also providing opportunities to further explore the mechanisms of globin gene regulation. In addition, animal models of β-hemoglobinopathies have demonstrated the feasibility of gene therapy for these conditions, now showing success in human clinical trials. Such models remain in use to dissect the molecular events of globin gene regulation and to identify novel treatments based upon the reactivation of developmentally silenced γ-globin. Here, we describe the development of animal models to investigate globin switching and the β-hemoglobinopathies, a field that has paralleled the emergence of modern molecular biology and clinical genetics.

Keywords: bacterial artificial chromosome; globin switching; green fluorescent protein; locus control region; sickle cell disease; β-Hemoglobinopathies.

Figure 1

Developmental expression of the β-like globins in humans and in WT and humanized transgenic mice. Notes: (A) Diagram of the human (upper) and mouse (lower) β-globin loci. Vertical bars represent Dnase I hypersensitive sites in the LCR. Embryonically expressed genes are shown in blue, fetal in green, and adult in red. Switching of the β-like globins in development is shown for (B) human, (C) mice, and (D) human β-like globins in transgenic mice. Values represent the proportion of total β-like globin transcripts detected in erythroid tissue. Note the dual switching events in humans, in contrast to the single mid-gestational switch in WT mice. Note also the mid-gestational switch of human γ- to β-globin expression in transgenic mice. Abbreviations: LCR, locus control region; WT, wild-type.

Figure 2

Visualization of sites of erythropoiesis using the ^Gγ-eGFP fluorescently tagged BAC reporter mouse. Notes: (A) The human β-globin locus, carried on a BAC, was modified via recombineering to replace the ^Gγ and ^Aγ genes with that of eGFP, under the control of the ^Gγ promoter. (B) Transgenic mouse embryos carrying the modified β-globin locus are shown under visible light (upper) and fluorescence illumination (lower). eGFP fluorescence marks the sites yolk sac blood islands at E7.5, the aorta-gonad-mesonephros and fetal liver at E10.5, and the fetal liver at E12.5 (arrows). Abbreviations: eGFP, enhanced green fluorescent protein; BAC, bacterial artificial chromosome.

Figure 3

Systemic iron accumulation in the Hbb^th-3/+ β-thalassemic mouse. Notes: Sections of spleen, liver, and kidney from WT or Hbb^th-3/+ heterozygous littermates were stained with Prussian blue to visualize iron deposits. The substantial presence of positive staining in the Hbb^th-3/+ samples, indicative of the dysregulated iron metabolism associated with the β-thalassemic phenotype (First described by Yang et al39). Abbreviation: WT, wild-type.

Figure 4

Recapitulation of IVSI-110 β-thalassemia splicing defect in BAC transgenic mice. Notes: (A) BAC recombineering was used to modify the 180-kb WT human β-globin locus so as to incorporate the IVSI-110 splicing mutation within the β-globin gene (B). HPLC analysis of globin profiles from (C) WT mice showed approximately equal proportions of murine α- and β-globin (^muα, ^muβ), whereas human β-globin made up approximately 10% of the total globins in WT mice carrying the native human β-globin locus (^huβ, shown in red) (D). (E) Heterozygous Hbb^th-3/+ mice carrying the WT human β-globin locus mice expressed higher levels of human β-globin, whereas (F) the presence of the IVSI-110 splicing mutation in the human β-globin locus abrogated expression substantially on the same β-thalassemic genetic background. Abbreviations: WT, wild-type; BAC, bacterial artificial chromosome.