HIGH-EFFICIENCY GENE-TRANSFER AND SELECTION OF HUMAN HEMATOPOIETIC PROGENITOR CELLS WITH A HYBRID EBV RETROVIRAL VECTOR EXPRESSING THE GREEN FLUORESCENCE PROTEIN/

We report a retroviral expression vector (PINCO) that allows high-effi ciency gene transfer and selection of hemopoietic progenitor cells (HP Cs), 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 encod es for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter, Transient transfection of PINCO in Phoenix p ackaging cells results in episomal propagation of the plasmid and gene rates viral titers as high as 10(7) 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 ran easily be monitored by fluorescent micr oscopy or fluorescence-activated cell-sorting (FAGS) analysis of livin g cells, The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation bet ween GFP and second gene expression levels in the infected cells, PINC O was used to infect human HPCs; infection efficiency was about 50%, G FP-positive cells can be FAGS sorted to yield a homogeneous population of infected cells, FACS-sorted GFP-positive HPC cells have, with resp ect to unfractionated HPC cells, the same frequency of long-term cultu re initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, fr om the transfection of the packaging cell line to the infection of tar get cells, requires less than a week, The high viral titer and the eas y obtainment of homogeneously infected cell populations without drug s election procedures make PINCO an ideal vector for gene transfer of hu man primary hemopoietic cells.