Humanized Mouse Model of Cooley's Anemia

Yongliang Huo,Sean C Mcconnell,Shan-Run Liu,Rui Yang,Ting-Ting Zhang,Chiao-Wang Sun,Li-Chen Wu,Thomas M Ryan

doi:10.1074/jbc.m805681200

Yongliang Huo, Sean C Mcconnell + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m805681200

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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.

Highlights

Hematological hallmarks of ␤ thalassemia are red blood cell microcytosis, hypochromia, targeting, and anisopoikilocytosis [2, 4]
Generation of the Cooley’s Anemia (CA) Mouse Model—Humans with CA are healthy at birth because of high HbF levels present in newborn circulating erythrocytes
The second murine ␤ thalassemia model was similar to the first, a deletion of ␤maj globin generated by targeted deletion in embryonic stem (ES) cells, but the phenotypes were different [14]

Summary

EXPERIMENTAL PROCEDURES

Targeting Construct and ES Cell Culture—Targeting plasmid, from 5Ј to 3Ј, contains 1.7 kb of mouse homology upstream of the mouse ␤maj globin gene (HindIII fragment), 5.7 kb human A␥ globin gene fragment (GenBankTM Accession No U01317: 38,066 – 43,728), 4.1 kb human ␤ globin gene fragment (GenBankTM Accession No U01317: 61,320 – 65,426), a phosphoglycerate kinase promoter driving a hygromycin resistance gene (hyg) flanked by two loxP sites, and 7 kb of mouse homology downstream of the mouse ␤min globin gene (BamHI fragment). The targeting plasmid was linearized by NotI digestion and electroporated into human ␥␤ KI hprt “tagged” ES cells3 [23]. 6-thioguanine (2 ␮M) was added to the ES medium 4 or 5 days after electroporation to select against cells that still contain the hprt “tagged” locus and enrich for homologous recombinants. DNA aliquots from ES cell colonies were screened by PCR to identify human ␥␤0 homologous recombinants. Hb concentrations were determined after conversion to cyanmethemoglobin by lysing red blood cells in Drabkin’s Reagent (Sigma), removal of insoluble erythrocyte membranes by centrifugation, measuring the absorbance at 540 nm on a spectrophotometer, and comparison to Hb standards. HPLC Analysis of Hemoglobin Chains—Hemolysates are prepared by lysing washed red blood cells in hemolysate buffer (5 mM phosphate, 0.5 mM EDTA, pH 7.4), adding NaCl to 1%, and removal of membranes by centrifugation.

Reverse primer

RESULTS

Wild type

DISCUSSION