The long-term aims of this project are to clarify the genetic control of hematopoietic differentiation and its disruption in leukemia. The focus for the next three years will be murine hematopoietic stem cells and the control of lineage commitment and maturation. Our pertinent recent studies have refined retroviral vectors that allow long-term expression in vivo, purified cycling hematopoietic stem cells, demonstrated the leukemogenic action of retroviruses bearing the bcr-abl, myc and Hox -2 .4 homeobox genes, discovered a homeobox gene (HLx) implicated in the control of myelomonocytic and lymphoid differentiation, and demonstrated that GATA transcription factors prompt megakaryocyte development. Our specific objectives are: (l) To assess the effects of immortalizing oncogenes on hematopoietic stem cells, with the aim of deriving long-term stem cell lines that remain multipotent and factor dependent. Our new retroviral vectors will be used to transduce genes that promote immortalization, namely myc, myb, bcl-2 and Hox-2.4, into purified stem cells derived from either normal mice or mice lacking the p53 tumor suppressor gene. The infected cells will be tested in vitro for extended clonal longevity and in vivo for ability to convey prolonged multilineage reconstitution. (2) To assess the biological effects on hematopoiesis of certain key transcriptional regulators. The function of factors associated with erythroid differentiation, notably GATA- l, EKLF and NF-E2, will be explored by introducing the genes into primitive hematopoietic cell lines and determining their phenotype and differentiation potential. Hlx functions in hematopoiesis will be evaluated by analysis of the perturbed lymphopoiesis found in Hlx transgenic mice and by the characterization of mice being made from recently generated embryonic stem cell lines having the Hlx gene disrupted. Justification. These studies should enhance to understanding of the nature of hematopoietic stem cells and the control of lineage commitment and maturation as well as improve retroviral gene delivery, issues pertinent to bone marrow transplantation and gene therapy. They should also clarify the normal role of certain proto-oncogenes and define early steps in leukemogenesis. As this work represents a logical extension of productive ongoing research, addresses central issues, exploits special resources and has an exceptional institutional base, it should substantially advance the molecular genetics of the hematopoietic system.
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