OuaSelect, a novel ouabain-resistant human marker gene that allows efficient cell selection within 48 h.
Efficient selection of gene-modified cells is required for a number of potential gene therapy applications, as well as molecular biology studies. Ideally, a clinical selection regimen would combine high selection speed, efficiency and efficacy, in addition to clinical grade selection techniques and low immunogenicity. To our knowledge, a selection marker satisfying all these features is so far not available. Ouabain is a clinically used cardiac glycoside and selective Na(+)/K(+)-ATPase inhibitor. On the basis of the high sensitivity of human Na(+)/K(+)-ATPase proteins to ouabain, and rapid killing of cells upon exposure, we have screened the ubiquitously expressed Na(+)/K(+)-ATPase alpha1 subunit for mutations that could greatly increase its resistance to ouabain. Two amino-acid substitutions, Q118R and N129D were sufficient to confer a two log greater resistance to ouabain in HeLa, Jurkat, U2OS cells and in primary cells. Furthermore, following transduction of primary lymphocytes with the alpha1(Q118R/N129D) gene, >99% pure populations of gene-modified cells were achieved with a recovery rate of >80% after 48 h of exposure to ouabain. These results identify the human alpha1(Q118R/N129D) (OuaSelect) as a promising selection marker gene for safe, rapid and cost-effective selection in clinical gene therapy and molecular biology research.
Treschow
A
A
Department of Medicine, Division of Hematology, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden.
Unger
C
C
Aints
A
A
Felldin
U
U
Aschan
J
J
Dilber
M S
MS
D000818:Animals; D002460:Cell Line; D004351:Drug Resistance; D005434:Flow Cytometry; D005818:Genetic Engineering; D005819:Genetic Markers; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010042:Ouabain; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D019076:Transgenes
D005819:Genetic Markers; D010042:Ouabain; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase