Enhanced ouabain resistance gene as a eukaryotic selection marker.
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-activated cell sorting requires complex equipment and may generate false-positive results when selection is performed 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 alpha(1) isoform of Na(+),K(+)-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 retrovirus 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.
Aints
Alar
A
Division of Hematology, Department of Medicine, Huddinge University Hospital, Karolinska Institute, SE-141 86 Stockholm, Sweden.
Belusa
Roger
R
Andersson
Ronnie M
RM
Güven
Hayrettin
H
Dilber
M Sirac
MS
D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004351:Drug Resistance / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007963:Leukocytes, Mononuclear; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics; D013552:Swine
D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase