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Green Fluorescent Protein as a Selectable Marker of Retrovirally Transduced Hematopoietic Progenitors

^a Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan;
^b Department of Cell Differentiation, Institute of Molecular Genetics and Embryology, Kumamoto University School of Medicine, Kumamoto, Japan;
^c Department of Hematology, Jichi Medical School, Tochigi, Japan

Key Words. Gene therapy • Green fluorescent protein • Hematopoietic progenitor • Retrovirus vector • Selectable marker • Flow cytometry

Dr. Akihiro Kume, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRa or CAG promoter. We then inserted an SRa-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo. Stem Cells 1999;17:226-232

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