ENHANCED GREEN FLUORESCENT PROTEIN AS SELECTABLE MARKER OF RETROVIRAL-MEDIATED GENE-TRANSFER IN IMMATURE HEMATOPOIETIC BONE-MARROW CELLS

The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduc ed cells, For this purpose, a retroviral vector was designed and teste d encoding a recombinant version of the Aequorea victoria green fluore scent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green flu orescence signal readily detectable by flow cytometry, Functional anal ysis of the retrovirally transduced bone marrow cells showed EGFP expr ession in in vitro clonogenic progenitors (GM-CFU), day 13 colony-form ing unit-spleen (CFU-S), and in peripheral blood cells and marrow repo pulating cells of transplanted mice. In conjunction with fluorescence- activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transd uced cells and to phenotypically define the transduced cells using ant ibodies directed against specific cell-surface antigens, Detrimental e ffects of EGFP expression were not observed: fluorescence intensity ap peared to be stable and hematopoietic cell growth was not, impaired. T he data show the feasibility of using EGFP as a convenient and rapid r eporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, an d to track these cells and ?heir progeny hath in vitro and in vivo. (C ) 1997 by The American Society of Hematology.