Humanized Mouse Model of Cooley's Anemia

Abstract

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

FIGURE 1.

Targeted gene replacement of adult mouse β globin genes with a human γ to β⁰ globin switching cassette. A, schematic demonstration of gene replacement in the mouse β globin locus tagged with human γβ globin and hprt genes with γβ⁰ globin and hyg genes. B, schematic demonstration of the wild-type mouse β globin locus and γβ⁰ KI allele after marker gene removal with labeled restriction endonuclease sites that confirm correct homologous recombination. The 5′ probe anneals to a 9.2-kb XbaI fragment from the wild-type allele and a 6.9-kb XbaI fragment from the human γβ⁰ globin KI allele. The 3′-probe derived from part of the β^min globin gene, anneals to a 14.8-kb EcoRI fragment from the β^min globin gene, a 7.3-kb fragment from the β^maj globin gene, and a 10.3-kb fragment from the γβ⁰ globin KI allele. C, Southern blot confirmation of mouse liver DNA of correct 5′ and 3′ homologous recombination. Lane 1, wild type control; lane 2, heterozygous γβ⁰ globin KI; lane 3, homozygous γβ⁰ globin KI.

FIGURE 2.

Determination of the mRNA splice variants (SV) from the human β⁰ globin KI allele caused by the IVS1.1 G to A mutation. A, alternatively spliced human β⁰ globin mRNA products were amplified by RT-PCR from peripheral blood RNA and resolved by PAGE. Lanes 1, 2, and 3 are the PCR products amplified from plasmid DNA from individually subcloned SV. Lane 4 is the PCR products amplified from γβ⁰ globin KI mouse. Lane 5 is the PCR product of wild-type human β globin. B, DNA sequence of each subcloned human β⁰ globin SV and wild type human β globin control. Two cryptic splice donor sites in the first exon (SV1 and SV2) and one cryptic splice donor site in the first intron (SV3) were used to process the mutant human β⁰ globin transcripts. C, schematic of the human β globin gene primary transcript illustrating exon/intron boundaries, locations of the primers used to amplify the SV, the G to A mutation of the first base of IVS1, and the approximate location of the cryptic splice donor sites.

FIGURE 3.

The humanγβ⁰ globin KI mice switch from mouse embryonic to human γ globin gene expression during fetal development. Circulating blood RNA from heterozygous (A) and homozygous (C)γβ⁰ globin KI embryos were analyzed by QPCR to quantify the level of β-like globin gene expression per gene copy during fetal development. Mouse embryonic βh1 (○) and εY (▵) globins are silenced by E14.5 and E16.5, respectively. Human γ globin (□) expression persists through fetal life becoming the predominant β-like globin chain produced in homozygous CA mice after E16.5. Already high at E14.5, mouse β globin (⋄) expression continues to rise into adulthood in heterozygous mice. Globin protein chains produced in heterozygous (B) and homozygous (D) γβ⁰ globin KI embryos during fetal development were measured by HPLC and expressed as a percentage of total β-like chains per gene copy. High levels of human γ globin chains are present throughout fetal life. n ≥ 3 for each time point in A–D. mβ is the average level of combined mouse β^maj and β^min globin gene RNA and protein levels. mεY and my are the mouse embryonic εY globin gene RNA and protein levels, respectively. hγ is the human γ globin gene RNA and protein levels. The low levels of short-lived hβ⁰ transcripts were left out of panels A and C for clarity, but may be found in supplemental Table S1.

FIGURE 4.

A, histology of blood, spleen, and liver, from heterozygous human γβ⁰ globin KI mice shows β thalassemic phenotypes. Wright-Giemsa stained peripheral blood smears of wild-type control and heterozygous γβ⁰ globin KI mice. Typical red blood cell targeting, hypochromia, and anisopoikilocytosis are observed in thalassemic human γβ⁰ globin KI mice. The normal red (erythroid) and white (lymphoid) pulp sections seen in the wild-type spleen were replaced by an expanded erythroid compartment in the γβ⁰ globin KI mice compared with the wild-type control. Livers of thalassemic γβ⁰ globin KI mice contain clusters of erythroid cells indicative of extramedullary hematopoiesis. Image magnifications are shown in parentheses. Slides were analyzed on Nikon Eclipse E800 or TE2000 microscopes (Nikon, Tokyo, Japan). Image magnifications are shown in parentheses. B, electron microscopy of circulating red blood cells from wild type and γβ⁰ globin KI adult mice demonstrating α globin chain inclusions (scale bar, 1 μm). C, comparison of spleen size as a percentage of body weight of wild type and γβ⁰ globin KI mice show a significant splenomegaly in the thalassemic mice (n ≥ 8 in each group).

FIGURE 5.

Humanized CA mice survive to birth with only human fetal hemoglobin in their red blood cells. A, homozygous newborn CA mice (right) are anemic as evidenced by the pale skin color compared with the heterozygous littermate control (left). B, peripheral blood smear of homozygous CA mice show a reduction in the absolute number of red blood cells that exhibit hypochromia, polychromatophilia, and anisopoikilocytosis all indicative of their severe anemia (100× magnification). C, HPLC chromatograms of peripheral blood hemolysates from newborn humanized mice. Control human HbA KI mice have peaks for human α globin and both human γ and β globin chains at birth (upper chromatogram). Homozygous CA KI mice have only human α and γ globin chains present in their red blood cells at birth confirming that the CA mouse survive solely upon human fetal Hb (lower chromatogram). D, histological sections of bone marrow and liver of wild type and homozygous CA newborn mice. The vascular lumens (asterisks) in the control bone marrow are filled with red blood cells while there is a paucity of red blood cells in the lumens of anemic CA mice.