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 ␥ to ␤ 0 globin gene cassette (␥␤ 0 ) was inserted directly into the murine ␤ globin locus replacing both adult mouse ␤ globin genes. The inserted human ␤ 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 ␤ globin polypeptide chains are produced. Heterozygous ␥␤ 0 mice suffer from microcytic anemia. Unlike previously described animal models of ␤ thalassemia major, homozygous ␥␤ 0 mice switch from mouse embryonic globin chains to human fetal ␥ globin during fetal life. When bred with human ␣ 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.

Human Globin Knock-in Mice Complete Fetal-to-Adult Hemoglobin Switching in Postnatal Development

“…Similarly, knock-in of this delayed-switching ␥␤ S cassette into the mouse ␤-globin locus results in the postnatal survival of the first humanized knock-in model of sickle cell anemia (50). Even in the absence of a functional ␤-globin gene, humanized ␥␤ 0 knock-in mice can survive to birth solely by synthesizing HbF (22). Interestingly, incorporation of hereditary persistence of fetal hemoglobin (HPFH) mutations into the ␥-globin promoter furthers extends the postnatal life of these humanized Cooley's anemia mouse models (21,23).…”

“…Heterozygous ␥␤ A knock-in mice were bred to cytomegalovirus (CMV)-Cre transgenic mice (30) to delete their loxP-flanked hygromycin marker gene. Human ␣2␣1-globin knock-in mice (22,23; T. M. Ryan, unpublished data) were bred to ␥␤ A knock-in mice to produce doubly heterozygous knock-in mice, which were interbred to obtain homozygous ␣2␣1/␣2␣1 ␥␤ A /␥␤ A knock-in mice (humanized mice) for analysis.…”

“…cDNA was prepared using the High Capacity cDNA kit (Applied Biosystems, Carlsbad, CA). Real-time PCR mixtures contained ABI TaqMan master mix (Applied Biosystems), 900 nM probe, 200 nM primers, and typically 1 to 10 ng of cDNA as described previously (22). PCRs for cDNA prepared from sorted cells were similar except that FastStart universal probe master mix (Roche Applied Science, Indianapolis, IN) was used.…”

“…At least four biological replicates were used for each qPCR experiment. Expression was calculated by the ⌬⌬C T method using experimentally determined primer efficiencies as described previously (22,34).…”

“…1C). In order to produce mice that synthesize complete human HbF and HbA, we bred the human ␥␤ A knock-in mice to animals with a similar targeted knock-in of the human ␣2-and ␣1-globin genes into the murine ␣-globin locus (22,23). Mice homozygous for both the human ␣2␣1 and ␥␤ A knock-ins expressed exclusively human hemoglobins within their definitive erythroid cells.…”