In vitro retroviral transfer of ras genes to single hemopoietic progenitors

Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.