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Erythropoietin and its receptor are required for definitive erythropoiesis and maturation of erythroid progenitor cells. Mice lacking the erythropoietin receptor exhibit severe anemia and die at about embryonic day 13.5. This phenotype can be rescued by the human erythropoietin receptor transgene. Animals expressing only the human erythropoietin receptor survived through adulthood with normal hematologic parameters and appeared to respond appropriately to induced anemic stress. In addition to restoration of erythropoiesis during development, the cardiac defect associated with embryos lacking the erythropoietin receptor was corrected and the increased apoptosis in fetal liver, heart, and brain in the erythropoietin receptor null phenotype was markedly reduced. These studies indicate that no species barrier exists between mouse and human erythropoietin receptor and that the human erythropoietin receptor transgene is able to provide specific expression in hematopoietic and other selected tissues to rescue erythropoiesis and other organ defects observed in the erythropoietin receptor null mouse.

Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.

Haptoglobin, a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma hemoglobin. Hemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of hemoglobin from the plasma and in the inhibition of glomerular filtration of hemoglobin. To evaluate these functions, Haptoglobin knockout (-/-) mice were created. These mice were viable but had a small, significant reduction in postnatal viability. Contrary to popular belief, the lack of haptoglobin did not impair clearance of free plasma hemoglobin in -/- mice. Induction of severe hemolysis by phenylhydrazine caused extensive hemoglobin precipitation in the renal tubular cells of both -/- and +/+ mice, with death occurring in 55% of -/- mice and in 18% of +/+ mice. In general, phenylhydrazine-treated -/- mice suffered greater tissue damage, as evidenced by the induction of hepatic acute phase response resulting in increased plasma alpha 1-acid glycoprotein (AGP) levels. Among -/- and +/+ mice that survived, -/- mice tend to suffer greater oxidative damage and failed to repair or regenerate damaged renal tissues, as indicated by their higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels and lower mitotic indices in their kidneys, respectively. This study suggested that a physiologically important role of hemoglobin-haptoglobin complex formation is the amelioration of tissue damages by hemoglobin-driven lipid peroxidation., (Copyright 1998 by The American Society of Hematology.)

Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of beta-versus alpha-globin chains, leading to the precipitation of excess alpha-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

Previous studies from our laboratories have shown that transgenic mice expressing high levels of beta S globin are well-protected from Plasmodium chabaudi adami and partially protected against P berghei (Shear et al, Blood 81:222, 1993). We have now infected transgenic mice expressing low (39%), intermediate (57%), and high (75%) levels of beta S with the virulent strain of P yoelii (17XL) that appears to cause cerebral malaria. We find that the level of protection in these three groups of mice correlates positively with the level of beta S chain expression in the mice. Seven of nine mice expressing the high level of beta S recovered from infection, as did 7 of 9 mice expressing the intermediate level of beta S. Control mice and mice expressing the lower level of beta S all succumbed to infection. In mice expressing high and intermediate levels of beta S, parasites were found almost exclusively in reticulocytes during recovery, suggesting that mature red blood cells expressing beta S are more resistant than reticulocytes. These studies confirm epidemiologic data and offer insight into the mechanism of protection of sickle trait individuals against falciparum malaria.

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.

All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma-positive cells) were used. Treatments with erythropoietin, 5-azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult.