Transcriptional activation of the granulocyte-macrophage colony-stimulating factor receptor gene in cell mutants

Retroviral insertional mutagenesis has proven to be a powerful in vivo appr oach for identifying genetic mutations involved in tumorigenesis or develop mental abnormalities. Applying this approach to an in vitro system, where e xperimental design can be readily manipulated, would greatly increase its e fficacy. In this study, we sought to determine whether retroviral insertion al mutagenesis could be used to isolate cell mutants, in which the transcri ptional activation of a receptor gene has occurred. Cells of the myeloid pr ogenitor cell line FDC-P1(M), which do not express the alpha receptor subun it (GMR alpha) for granulocyte-macrophage colony-stimulating factor (GM-CSF ), were infected and selected for growth in GM-CSF. Over 100 mutants were i solated at a frequency up to ninefold higher than that of uninfected contro ls. Expression of GMR alpha in these mutants was confirmed by blocking prol iferation with GM-CSF antibodies, detection of high-affinity receptors, and Northern blot analysis. Significantly, in 7/18 mutants analyzed, gross DNA rearrangements had occurred in the GMR alpha locus. These rearrangements w ere demonstrated to be due to intergenic rearrangements, juxtaposing an act ive enhancer/promoter upstream of the GMR alpha gene. In one mutant it coul d be demonstrated that the wild-type allele was also expressed, providing e vidence that secondary mutations had occurred. The implications of these re sults for retroviral insertional mutagenesis are discussed. (C) 2000 Academ ic Press.