LONG-TERM EXPRESSION OF THE GENE ENCODING GREEN FLUORESCENT PROTEIN IN MURINE HEMATOPOIETIC-CELLS USING RETROVIRAL GENE-TRANSFER

Background. A major goal in retroviral-based gene therapy is to establ ish methods that allow for selection and tracking of transduced cell p opulations. Green fluorescent protein (GFP) may be useful for gene the rapy applications because it is a naturally fluorescent protein that c an be detected using conventional flow cytometers facilitating rapid a nalysis and purification of transduced cell populations. However, it i s unknown whether GFP can be stably expressed in vivo, particularly in multiple bone marrow-derived cell lineages, Methods. A murine retrovi rus carrying the gene encoding GFP was used to infect murine bone marr ow cells (BMCs), These studies were conducted to (1) directly determin e whether GFP could be used as a marker of BMC transduction, (2) deter mine whether GFP is capable of being expressed in multiple bone marrow -derived hematopoietic cell lineages, and (3) determine whether GFP co uld be used to follow the fate of transduced cells in vivo. Results. I nfection of BMCs with retroviruses carrying the gene encoding GFP resu lted in a fluorescent signal in viable transduced cells that was detec table by flow cytometry, Expression of GFP was detected in multiple bo ne marrow-derived cell lineages after transduction, including stem cel l antigen-positive (Sca-1(+)), lineage marker-negative (Lin(-)) cells. Using GFP as a selectable marker, we were able to enrich for transduc ed cells by cell sorting. Mice reconstituted with enriched populations of GFP(+) cells showed a significant increase in the percentage of ce lls expressing GFP in the periphery when compared with mice reconstitu ted with unenriched transduced bone marrow. Conclusions. These data in dicate that GFP can be used to select for transduced BMCs in vitro, ex pressed in multiple bone marrow-derived cell lineages, used to select transduced cells, and follow the fate of transduced cells long-term in vivo.