Beta-thalassemic mice will be used to study the regulation of exogenous mouse and human beta-globin genes in transfected and transplanted bone marrow stem cells. For these studies, two congenic strains of beta-thalassemic mice are being developed that differ from the C57BL/6 and DBA/2J inbred partners at the Hbb locus. The hematological parameters of these mice will be determined so the restorative potential of various bone marrow therapies can be assessed. These will include peripheral blood hematology, red cell survival, red cell production, and the sizes of the CFU-S, CFU-C, BFU-E and CFU-E pools in the hematopoietic tissues. Various combinations of numbers of marrow cells injected and pretreatment of recipients with X-rays and chemicals will be investigated to find an optimal and/or the most practical condition to promote long-term survival. Using these conditions, mixtures of normal and beta-thalassemic marrow cells will be injected into the same recipient to assess the relative potential of normal (as a model for """"""""genetically repaired"""""""") stem cells to establish functional grafts in the presence of the host's marrow and environment. The above studies will establish the protocols to be used to investigate the effects extra beta-globin genes may have on bone marrow function. Bone marrow cells from BALB/c mice and a BALB/c mutant that carries an extra beta-globin gene complex will be transfused into lethally irradiated BALB/c mice to investigate whether the duplicated segment containing Hbb affects stem cell function. The number of CFU-S and duration of recovery obtained from marrow cells incubated for 24 to 48 hours under normal and transfecting culture conditions will be compared to determine the effect of transfection per se. DNA from spleen colonies derived from single CFU-S will be analyzed by Southern blotting to determine the number of stem cells transfected. The level of expression of the exogenous beta-globin genes in hematopoietic tissues of long-term survivors will be determined by quantitation of the gene-specific protein or mRNA synthesis. Bone marrow from 2- and 6-month survivors will be retransplanted into lethally irradiated mice and the DNA from individual spleen colonies will be analyzed by Southern blotting to establish whether the insertions appear in fully restored marrow. A S-phase specific drug, 6-thioguanine, will be used to investigate the proportion of nondividing Go and cycling CFU-S cells that become transfected and retain the exogenous beta-globin genes for 2 to 6 months.
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| Ampel, N M; Van Wyck, D B; Aguirre, M L et al. (1989) Resistance to infection in murine beta-thalassemia. Infect Immun 57:1011-7 |
| Popp, R A; Bolch, S L; Shinpock, S G et al. (1989) Expression of the globin genes and hematopoiesis in beta-thalassemic mice. Adv Exp Med Biol 271:161-76 |
| Bolch, S L; Shinpock, S G; Wawrzyniak, C J et al. (1989) A comparison of stem cell populations and hemoglobin switching in normal versus beta-thalassemic mice. Exp Hematol 17:340-3 |
