Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines.

Retroviral vectors are widely used for the introduction of exogenous genetic material into hematopoietic cells. Here we report the generation of retroviral vectors containing the Aequorea victoria green fluorescent protein (GFP) gene and improved versions thereof. Murine fibroblasts transduced with the mutant GFP genes demonstrated a distinct green fluorescent signal in fluorescence-activated cell sorter (FACS) analysis. The relative intensities of peak green fluorescence observed with different GFP mutants were in the order EGFP>hGFP(S65T)> GFP-PTS1 or RSGFP>wildtype GFP (wtGFP). Furthermore, GFP-PTS1 expression was observed in murine (3T3, Rat2, and freshly-cultured bone marrow) and human (K562) cells transduced with the corresponding retroviral vector. The GFP-PTS1 positive phenotype could be selected for by FACS and appeared to be stable for at least 1 month in murine fibroblasts and human K562 cells. Therefore, these GFP variants are convenient selectable markers to monitor retroviral-mediated gene transfer and expression in mammalian hematopoietic cells.