The in-vivo proliferative and self-renewal kinetics of murine pluripotent hematopoietic stem cells (CFU-S) will be studied using retroviral mediated gene transfer. The techniques of recombinant vectors, molecular biology and cell biology will be employed to study key hypotheses pertaining to hematopoietic stem cells. In addition, the use of a recombinant retrovirus that transfers a selectable marker will be developed and used to attempt in-vivo selection of murine hematopoietic stem cells.
Specific aims of these studies will be: 1) to determine the in-vivo proliferative kinetics of transduced murine pluripotent hematopoietic stem cells (CFU-S) in a fully reconstituted transplant recipient following retroviral-mediated gene transfer; 2) to measure self-renewal capacity of differentiating and non-differentiating transduced murine CFU-S using retroviral-mediated gene transfer to establish trackable CFU-S clones in the intact mouse. The hypothesis of structured variation in proliferative kinetics of stem cell populations will be tested; 3) to construct a recombinant retroviral vector containing a dihydrofolate reductase (DHFR) cDNA that encodes a mutant enzyme with a lowered methotrexate (MTX) affinity; 4) to attempt to select, using MTX, transduced hematopoietic stem cells (CFU-S) in vivo and hematopoietic progenitors (BFU-E, CFU-GM) in vitro after gene transfer using the DHFR recombinant retroviral vector. The information obtained by these studies will enhance our basic understanding of stem cell kinetics as well as provide rational approaches for the use of retroviral mediated gene transfer to correct severe genetic disease of the hematopoietic system.
