These studies will extend technologies of genetic-marking to an analysis of hematopoietic stem cell clonal behavior in normal non-ablated animals. Such a fate-map of the normal unperturbed hematopoietic system in comparison to ongoing studies utilizing irradiated hosts for stem cell assays will provide a first assessment of cell intrinsic vs. environmental factors in the formation of all blood cell lineages in normal as well as pathological conditions such as leukemia. In addition, these studies should shed light on parameters involved in bone marrow transplantation therapies. In the first strategy, purified retrovirally-marked stem cells will be engrafted into normal and mutant fetal mice by transplacental injection. Such cells should subsequently colonize the bone environment in a normal fashion and at the correct developmental time and contribute to hematopoiesis throughout adult life. A second strategy will combine germ- line transgenesis with somatic retroviral-mediated gene transfer to generate lines of mice that will introduce random stable proviral markers into hematopoietic stem cells as they develop in the normal environment. The key principal is the germ-line introduction of chimeric retroviral transgenes whose expression is targeted to cells comprising the niches within which hematopoietic stem cells proliferate. This will result in local production of marker virus which will infect the stem cells, inactivate itself local production of marker virus which will infect he stem cells, inactivate itself and therefore uniquely and permanently mark the cells and their resultant clonal progeny. The expression of marker virus is designed to occur only in the temporal frame during which stem cells first appear and clonally expand during ontogeny. Sequential and quantitative Southern blot analysis of proviral marker distribution in peripheral blood cell types obtained from animals derived from both approaches will define the developmental and proliferative behavior of the stem cell in its normal environment. The second proposed set of experiments will provide inroads to a precise molecular dissection of regulatory mechanisms which function in proliferation/differentiation decisions of primitive stem cells. Strategies are proposed to analyze gene expression directly in purified stem cells and to isolate stem cell specific members of regulatory gene families. These will provide direct insight into hematopoietic regulation as it functions in normal blood formation and malfunction to yield cancerous or other hematologic disorders.
Showing the most recent 10 out of 11 publications
