The overall goals of the proposed work continue to be (i) the development of efficient methods for genetically modifying hematopoietic stem cells, (ii) the development of suitable methods for obtaining the appropriate in vivo expression of transferred genes, and (iii) the development of experimental model systems useful for understanding the control of hematopoiesis and for assessing the feasibility of genetic therapies for diseases affecting the hematopoietic system. The work currently proposed focuses in a major way upon the use of purified murine hematopoietic stem cells, rather than unfractionated bone marrow cells, as targets for gene transfer. An important component of the proposed work involves the physical and functional characterization of purified hematopoietic stem cells, with a particular emphasis upon understanding the functional properties of different hematopoietic cell populations that are important either for efficient genetic modification of stem cells or for the successful engraftment of lethally irradiated recipients with genetically modified cells. The proposed gene expression studies focus primarily upon features of retrovirus vector design and features of the inserted sequences themselves which influence the expression of recombinant retroviral genomes in vivo. An important new goal of this research program is to determine the feasibility of using homologous recombination techniques to 'repair' defective genes present in hematopoietic stem cells.