RETROVIRAL-MEDIATED TRANSFER OF THE GREEN FLUORESCENT PROTEIN GENE INTO MURINE HEMATOPOIETIC-CELLS FACILITATES SCORING AND SELECTION OF TRANSDUCED PROGENITORS IN-VITRO AND IDENTIFICATION OF GENETICALLY-MODIFIED CELLS IN-VIVO

We have investigated the utility of the green fluorescent protein (GFP ) to serve as a marker to assess retroviral gene transfer into hematop oietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a nat urally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cy tochemical staining to detect its expression. A bicistronic murine ste m cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-t iter, ecotropic retroviral producer cells free of replication competen t virus were generated and used to transduce murine bane marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were in tensely fluorescent compared to mock-transduced cells or cells transdu ced with a control retrovirus. Erythroid and myeloid hematopoietic col onies derived from GFP-transduced marrow were easily scored for retrov iral gene transfer by direct in situ fluorescence microscopy. Clonogen ic progenitors expressing increased levels of antifolate drug resistan ce could be enriched from the GFP-transduced marrow population by fluo rescence activated cell sorting of cells expressing high levels of GFP . In vivo, splenic hematopoietic colonies and peripheral blood cells f rom animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for s coring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high le vels of the transgene. (C) 1997 by The American Society of Hematology.