HUMAN GENE THERAPY 13:969–977 (May 20, 2002) © Mary Ann Liebert, Inc. Enhanced Ouabain Resistance Gene as a Eukaryotic Selection Marker ALAR AINTS, 1 ROGER BELUSA, 2 RONNIE M. ANDERSSON, 2 HAYRETTIN GÜVEN, 1 and M. SIRAC DILBER 1 ABSTRACT Current selection markers allow selection by antibiotics or fluorescent/magnetic sorting by green fluorescent protein or membrane antigens. Antibiotic selection proceeds on a time scale of weeks, and flourescence-acti- vated cell sorting requires complex equipment and may generate false-positive results when selection is per- formed too early after transduction with membrane markers. We have characterized an endogenous eukaryotic selection marker, the ouabain resistance gene (Oua r ), which has the potential for quick and efficient in vitro selection of target cells. The Oua r used by us is derived from the rat a 1 isoform of Na 1 ,K 1 -ATPase, where leucine at position 799 is substituted for cysteine by targeted mutagenesis. This mutation confers resistance to more than 1 mM ouabain in vitro. We show that cells transfected with plasmid or transduced with a retro- virus vector encoding Oua r can be selected efficiently with ouabain in 48 hr and that a pure population of cells can be obtained. The ouabain resistance gene may be useful as a selection marker in general molecular biology, preclinical, and clinical applications because of its short selection time and also because of the safety of ouabain for human use. 969 OVERVIEW SUMMARY We have developed a new and efficient eukaryotic selection marker for human use from rat Na 1 ,K 1 -ATPase a 1 sub- unit. The mutation used by us, L799C, confers resistance to more than 1000 mM ouabain. The selection process relies on particularly rapid elimination of human cells by a car- diac glucoside, ouabain, via inhibition of ion transport across the plasma membrane. The selection process is not genotoxic. We present successful genetic modification and selection of both cell lines as well as primary blood mononu- clear cells. The human cells can be modified transiently by transfection, or stably by retroviral transduction, and se- lected in 48 hr to obtain a pure population of gene-modi- fied cells. The method is useful in general molecular and cell biology applications as well as in gene therapy applica- tions for preclinical and clinical selection of human cells. INTRODUCTION G ENETIC LABELING AND SELECTION of cells has been the key issue in cell biology applications. Current gene therapy vectors are limited by various issues, including low gene trans- fer efficiency and requirement for extended in vitro cell culture for selection. To optimize gene transfer into human cells, two approaches have been taken. The first approach is to improve gene delivery vehicles; the second approach is to improve ex vivo culture conditions to increase the gene transfer efficiency while preserving the developmental and physiologic properties of the modified cells. An important factor in the process is the selection time. Many primary cells, most notably early hema- topoieticprogenitorcells, respond to in vitro cell culture by dif- ferentiation and losing pluripotency. Also, cytotoxic T cells have been shown to lose activity after prolonged in vitro se- lection (Carlens et al., 2002). Thus it would be advantageous 1 Division of Hematology, Department of Medicine, Huddinge University Hospital, Karolinska Institute, SE-141 86 Stockholm, Sweden. 2 Pediatric Unit, Department of Woman and Child Health, Astrid Lindgren Children’s Hospital Research Laboratory, Karolinska Institute, SE- 171 76 Stockholm, Sweden.