Diana M. Popp

A mouse model for β-thalassemia

Abstract A mutation that produces an absolute deficiency of normal β-major globin polypeptides has been recovered from a DBA/2J male mouse. Most mice homozygous for the deficiency survived to adulthood and reproduced but were smaller at birth than their littermates and demonstrated a hypochromic, microcytic anemia with severe anisocytosis, poikilocytosis, and reticulocytosis and the presence of inclusion bodies in a high proportion of circulating erythrocytes. Mice heterozygous for the deficiency demonstrated a mild reticulocytosis but were not clinically anemic. Analysis of globin chain synthesis in vitro by 3 H-leucine incorporation revealed that β-globin synthesis was nearly normal (95%) in heterozygotes and about 75% of normal in deficiency homozygotes. Molecular characterization of the mutation by restriction analysis revealed a deletion of about 3.3 kb of DNA, including regulatory sequences and all coding blocks for β-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbb th-1 , represent the first animal model of β-thalassemia (Cooleys anemia), a severe genetic disease of humans.

DNA repair in a congeneic pair of mice with different longevities.

The rate of ultraviolet light (UV)-induced DNA excision repair was determined in embryonic cells derived from a congeneic pair of short-lived (C57BL/10.F) and long-lived (C57BL/10) mice. Excision repair was measured by both bromodeoxyuridine photolysis and arabinofuranosyl cytosine inhibition. No difference in rate of repair was observed between the two cell lines.

Hematology of a murine beta-thalassemia: a longitudinal study.

Mice homozygous for a spontaneous mutation, in which the beta-major globin gene is deleted, have clinical symptoms of beta-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in beta-thalassemic mice are similar to those observed in human beta-thalassemia. Synthesis of beta-globin is depressed but not as much as might be expected because the expression of the beta-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged pool of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous beta-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects of newly developed iron chelators.

Basal serum immunoglobulin levels

Two congenic strains of mice were identified that differ in their serum immunoglobulin levels. The strains were crossed, the F1 progeny were intercrossed, and the serum immunoglobulin levels of the F1 and F2 progeny were analyzed. The F1 mice have serum immunoglobulin levels like that of the high parent, and the low-immunoglobulin phenotype segregates in the F2 population. Six other inbred strains of mice were also characterized for basal serum levels of five classes of immunoglobulin.

An analysis of genetic factors regulating life-span in congeneic mice

Controlled matings between two strains of mice that have minimum genetic heterogeneity but whose mean ages at death are significantly different were used to identify genetic factors that govern longevity. The congeneic pair, C57BL/10 (B10) and C57BL/10.F (B10.F), differ at a region of the genome in and around the H-2 complex and have mean ages at death of 706 +/- 14 and 456 +/- 15 days, respectively. B10.F also has a reduced level of basal serum IgA levels, a trait which segregates in F2 progeny. F2 and back-cross progeny were classified for the H-2 genotype and allowed to live out their life-span. Survival curves for F2 and backcross progeny selected on the basis of their H-2 type show that the progeny homozygous for the H-2 allele of B10.F have a mean age at death significantly different and reduced from that of the progeny homozygous for the H-2 allele of B10.

Hemopoietic stem cell heterogeneity: use of cell cycle-specific drugs to look for age-associated alterations.

Hemopoietic tissue is vulnerable to perturbations, and data show that it is an appropriate tissue in which to look for age-associated alterations. This tissue has a high regenerative capacity, is composed of a heterogeneous population of stem cells that are capable of self renewal or differentiation, or both, and is sustained by a pool of resting cells. The heterogeneity of bone marrow has made characterization of the cellular elements difficult. Techniques commonly used to identify and quantify the various maturation levels of hemopoietic stem cells and the limitations of these techniques are discussed. Most techniques used to assay age-associated changes in bone marrow have not differentiated between specific cellular alterations or shifts in the distribution of the cellular elements. In particular, it has been difficult to determine the stability of the non-dividing stem cell because of the low incidence of this cell (6 per 1000) and the lack of a specific assay for this important cell type. The use of cell cycle-specific drugs has provided quantitative information on specific subpopulations of hemopoietic stem cells and seems to be the most promising approach towards determining qualitative and quantitative differences in the hemopoietic stem cells of young and old individuals.

Expression of the Globin Genes and Hematopoiesis in Beta-Thalassemic Mice

Mice homozygous for a deletion of the beta-dmajor globin gene exhibit clinical symptoms of human beta-thalassemia and are good experimental animals for investigating the regulation of globin gene expression, perturbation of hematopoiesis, and potential methods for treating patients with beta-thalassemia. Homozygous beta-thalassemic mice have a microcytic anemia, their red blood cells display anisocytosis, poikilocytosis and a shortened life span, and iron overloading occurs in several tissues in response to increased erythropoiesis2,3. Mice heterozygous for the beta-thalassemia1,2 mutation are clinically normal.

RELATIVE ABILITY OF PARENTAL MARROWS TO RE-POPULATE LETHALLY IRRADIATED F1 HYBRIDS

The relative abilities of C57BL and 101 marrow to repopulate the hematopoietic system of lethally x-irradiated mice was determined. The disproportionately greater ability of 101 than C57BL cells to repopulate (C57BL X 101) F/sub 1/ recipients is reported, and data are given on the relative ability of marrow from other strains of mice to repopulate lethally irradiated mice. Consistent with the observation that C57BL marrow is less efficient that 101 marrow is the finding that the number of C57BL cells required to produce functioral long-terra marrow grafts was much larger than the number of 101 cells. Factors that might influence the differential in the growth ability of the two types of marrow are discussed. (H.M.G.)

Peroxidase staining combined with autoradiography for study of eosinophilic granules.

Giemsa staining and a peroxidase reaction were applied to blood films in conjunction with autoradiography to establish the types of granulocytes that stain differentially with the benzidine-peroxidase reaction. Differential counts made on Ciemsa-stained and peroxidase-stained autoradiograms were compared. In T. spiralis-infected rats with an elevated eosinophil count, as judged by Giemsa staining, the percentage of granulocytes that stained more intensely with peroxidase was increased. The results suggested that the eosinophils were the intensely peroxidase-positive cells. Blood smears were stained for peroxidase before being coated with NTB2 liquid emulsion. Although the blue color of the peroxidase reaction faded during photographic development, the color redeveloped when peroxidase-stained autoradiograms were stained once again after photographic development. It was found necessary to stain for peroxidase both before and after autoradiography. The correlation of Giemsa-stained and peroxidase-stained au...

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation.

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781‐788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross‐linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild‐type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A‐activated receptors can stimulate mitogen‐activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S‐phase are both decreased relative to 1 nM wild‐type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild‐type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waals weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival. J. Cell. Biochem. 83: 326–341, 2001.