The establishment of hematopoiesis in culture from murine embryonic stem (ES) cells provides a powerful approach for studying early commitment steps as hematopoietic precursors develop from pre-hematopoietic mesoderm. Analysis of developing embryoid bodies has demonstrated that hematopoietic differentiation in these cultures recapitulates many aspects of the onset of hematopoiesis in utero, including vasculogenesis and the switch from the primitive to the definitive program. We previously used CCE ES cells transduced with our MSCV retroviral vector overexpressing the diverged homeobox gene HOX11 of T-cell acute lymphoblastic leukemia to establish factor-dependent hematopoietic precursor cell lines arrested at novel stages of primitive and definitive hematopoietic development. These results, demonstrating proof-of-concept, provide the rationale for the present proposal to extend this strategy to obtain conditionally-blocked murine hematopoietic precursor cell lines with a variety of differentiative potentials. We are particularly interested in the possibility of conditionally arresting common endothelial/hematopoietic precursors (i.e., hemangioblasts) as well as hematopoietic stem cells with long- term in vivo repopulating activity. To this end, our specific aims are to develop two systems for conditional immortalization: the first, based on site-specific excision of HOX11 by adenovirus-mediated transient expression of Cre recombinase; and the second, based on a tetracycline-regulatable HOX11 retroviral vector. While both approaches will permit precursor-progeny relationships to be unequivocally established, we hypothesize that the latter strategy will allow clonal descendants of precursor cells to be arrested and amplified at successive stages of the hematopoietic hierarchy. It is envisioned that the multipotent precursor lines obtained will have broad utility for molecular biological investigations of hematopoietic differentiative progression and lineage restriction. Toward this goal, homogenous populations of clonally-related precursor cells will be used as starting material for identification by cDNA microarray technology of differentially expressed genes as candidate regulators of hematopoietic cell fate decisions. It is anticipated that the information gained from these studies will also provide insight into the potential roles of those homeobox genes implicated in the transcriptional control of hematopoiesis as well as lead to a better understanding of the underlying mechanism of leukemia initiation mediated by HOX11. Successful realization of these goals will provide a basis for future research endeavors involving primate ES cells.
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