The overall objective of this project is to determine if defective Friend spleen focus-forming virus (SFFV) carries genetic information which is able to function as a hemopoietic self-renewal gene when introduced into the pluripotent hemoietic stem cell (CFU-S) of murine bone marrow cells.
Major Specific Aims; SFFV constructions. To construct a transmissible retrovirus consisting of the molecularly cloned Friend SFFV and an altered mouse dihydrofolate reductase (dhfr) sequence. The presence of a single mutant dhfr sequence serves as a dominant-acting selectable gene. Persistence if engineered defective SFFV. Suspensions of murine bone marrow cells enriched for CFU-S will be exposed to infectious SFFV-dhfr to determine if the host range of this engineered, defective virus includes hemopoietic cells. Virus-treated marrow will be injected into lethally-irradaited histocompatible adult recipients. These mice will be monitored for latent SFFV persistence. SFFV and stem cells self-renewal. Using donor marrow which are predominantly SFFV+ CFU-S, to probe for SFFV-induced alterations in host hematopoiesis and stem cell renewal. SFFV+ marrow cells will be retrieved from latently infected mice and serially transplanted to determine their ability to self-renew and protect or rescue lethally-irradiated recipients from total hemopoietic failure and death. To insure repopulation by predominantly SFFV+ CFU-S, we shall treat reconstituted mice with a drug regimen if methotrexate (Mtx). Identify of an SFFV self-renewal gene. To determine if the envelope (env) sequences which encode gp52sfv are required for the induction of extended CFU-S self renewal. A number of well-defined SFFV insertion-deletion mutants will be reconstituted with dhfr, pseudotypes with helper, and used to infect CFU-S. Latent persistence will be confirmed using an SFFV representative probe, while the SFFV+ stem cells will be monitored for self-renewal capacity via serial transplantation into lethally irradiated syngeneic adult mice. Vector transfer of self-renewal may eventually be exploited to the benefit of the host whether he/she be suffering from an aquired or congenital defect in hemopoiesis erytghromyeloid and lymphoid.
| Eckner, R J; Bennett, M; Hettrick, K L et al. (1987) Natural killer cell suppression of Friend virus-induced preleukemic hemopoietic stem cells. J Virol 61:2631-8 |
