HIGH-TITER RETROVIRAL VECTORS CONTAINING THE ENHANCED GREEN FLUORESCENT PROTEIN GENE FOR EFFICIENT EXPRESSION IN HEMATOPOIETIC-CELLS

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a pro ducer cell line that yields high titers of amphotropic retroviral vect ors carrying the enhanced green fluorescent protein (EGFP) gene, a cod on humanized, red-shifted variant of the green fluorescent protein (GF P) gene, which can be used as a selectable marker. We have used a hybr id vector that has been shown to efficiently drive gene expression in hematopoietic cells, Virtually all murine and human cell lines and pri mary human hematopoietic cells tested were transduced with varying eff iciency after incubation with vector-containing supernatants, Human CD 34(+) cells obtained front cord blood or aphaereses products were tran sduced using a protocol that involves daily addition of vector-contain ing supernatants for 6 consecutive days, At day 6, up to 16% of the ce lls expressed EGFP, as assessed by flow cytometry. Sorted EGFP-express ing cells were able to produce fluorescent hematopoietic colonies. EGF P's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transdu ction efficiency along with other phenotypic markers. (C) 1997 by The American Society of Hematology.