Humanized mouse models of Cooley's anemia: correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology.

beta thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation.

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing-NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin beta-chain (betaCys93) and, specifically, for S-nitrosation of betaCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the betaCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell-mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of betaCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell-dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo.

Differential binding of erythroid Krupple-like factor to embryonic/fetal globin gene promoters during development.

The competition model for beta-like globin gene switching during development predicts that differential binding of transcription factors to globin gene promoters and/or proximal enhancers regulate the competitive interactions of globin gene family members with the powerful locus control region (LCR). Direct interactions of individual genes with the LCR are essential for high level expression in erythroid cells. In this paper, we have demonstrated, by chromatin immunoprecipitation, that erythroid-Krupple-like factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive (but not in definitive) erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2, and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is 3-fold higher than the level in primitive cells. These results suggest that temporal-specific changes in EKLF abundance result in differential binding of this essential erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR.

Correction of sickle cell disease by homologous recombination in embryonic stem cells.

Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (beta(S)) with a normal copy of the gene (beta(A)). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human beta(S)-globin gene with a human beta(A)-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.

The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.