¹Division of Genetic Therapeutics, Center for Molecular Medicine, Tochigi, Japan

²CREST, Japan Science and Technology Corporation, Tochigi, Japan

³DNAVEC Research Inc., Ibaraki, Japan

⁴Department of Hematology, Jichi Medical School, Tochigi, Japan

Correspondence to: A Kume, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation. Gene Therapy (2000) 7, 1193-1199.

gene therapy; hematopoietic stem cells; green fluorescent protein; CD24; bone marrow transplantation; flow cytometry