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 g enetic material into hematopoietic cells. Here we report the generatio n of retroviral vectors containing the Aequorea victoria green fluores cent protein (GFP) gene and improved versions thereof. Murine fibrobla sts transduced with the mutant GFP genes demonstrated a distinct green fluorescent signal in fluorescence-activated cell sorter (FACS) analy sis. The relative intensities of peak green fluorescence observed with different GFP mutants were in the order EGFP>hGFP(S65T)>GFP-PTS1 or R SGFP>wildtype GFP (wtGFP). Furthermore, GFP-PTS1 expression was observ ed in murine (3T3, Rat2, and freshly-cultured bone marrow) and human ( K562) cells transduced with the corresponding retroviral vector. The G FP-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 marker s to monitor retroviral-mediated gene transfer and expression in mamma lian hematopoietic cells. (C) 1997 Academic Press.