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[Cancer Research 58, 14-19, January 1, 1998]
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High-Efficiency Gene Transfer and Selection of Human Hematopoietic Progenitor Cells with a Hybrid EBV/Retroviral Vector Expressing the Green Fluorescence Protein1

Francesco Grignani, Todd Kinsella, Amedea Mencarelli, Mauro Valtieri, Daniela Riganelli, Fausto Grignani, Luisa Lanfrancone, Cesare Peschle, Garry P. Nolan and Pier Giuseppe Pelicci2

Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy [P. G. P., L. L.]; Istituto di Medicina Interna e Scienze Oncologiche, Perugia University, 06100 Perugia, Italy [Fr. G., A. M., D. R., Fa. G., P. G. P.]; Hematology Oncology Laboratory, Istituto Superiore di Sanità 00161 Rome, Italy [Fr. G., M. V., C. P.]; Department of Molecular Pharmacology, Stanford University, Stanford, California 94305 [T. K., G. P. N.]; and Thomas Jefferson University, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107 [C. P.]

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 107 colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

1 Supported by Italy-USA Therapy of Tumors, ISS (Rome, Italy).

2 To whom requests for reprints should be addressed, at Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti, 435, 20141 Milan, Italy. Phone: 39-2-574-89-831; Fax: 39-2-574-89-851; E-mail: pgpelicci@ieo.cilea.it.

Received 10/22/97. Accepted 11/13/97.




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