DYSREGULATED EXPRESSION OF GATA-1 FOLLOWING RETROVIRUS-MEDIATED GENE-TRANSFER INTO MURINE HEMATOPOIETIC STEM-CELLS INCREASES ERYTHROPOIESIS

Retrovirus-mediated gene transfer was used to study the effects of dys regulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral v ector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GAT A-1 was demonstrated by super-shift analysis with a monoclonal antibod y against murine GATA-1 using extracts of nonerythroid cytotoxic T-lym phocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recip ient animals. Polymerase chain reaction (PCR) analysis performed on DN A extracted from peripheral blood 12 to 40 weeks posttransplantation d emonstrated the presence of the PGK-GATA-1 provirus. Proviral integrit y and copy number were demonstrated by Southern blot analysis of DNA f rom spleen, thymus, and bone marrow tissues from the long-term animals . At 16 weeks posttransplant, animals that received cells transduced b y the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) cou nt than control animals that received cells transduced with a Virus co ntaining a neo' gene. Erythropoiesis was stimulated in GATA-1 and cont rol animals by phlebotomy. GATA-1 animals required more extensive phle botomy to reach a hematocrit less than 25 and their hematocrit returne d to normal levels sooner than control animals. The affect of twice-da ily injections of 10 U recombinant erythropoietin (epo) was also exami ned. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data su ggest that dysregulated expression of GATA-1 in primitive hematopoieti c cells enlarges the pool of epo-responsive erythroid progenitor cells . (C) 1995 by The American Society of Hematology.