Generation of a Novel Mouse Model of Cooley’s Anemia.

A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β⁰ thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human ^Aγ-globin gene, 4.1kb human β⁰-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β⁰-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β^0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β^0-IVS1(GtoA)-globin gene mutation is a naturally occurring β⁰ thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ⁰-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β⁰ thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α₂γ₂) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β⁰-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.