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OuaSelect, a novel ouabain-resistant human marker gene that allows efficient cell selection within 48 h

Gene Therapy volume 14, pages 1564–1572 (2007)Cite this article

Abstract

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 α1 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 α1Q118R/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 α1Q118R/N129D (OuaSelect) as a promising selection marker gene for safe, rapid and cost-effective selection in clinical gene therapy and molecular biology research.

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Acknowledgements

We are grateful to Birger Christensson (Centre for Cell analysis at Karolinska University Hospital Huddinge) for help with confocal images, CI Edvard Smith for editorial assistance, and Hernan Concha and Abdalla M Jama, for their technical assistance. This work was supported by grants from (1) EU Commission (QLK3-CT-2001-01265), (2) Petrus och Augusta Hedlunds Stiftelse and (3) The Swedish Cancer Society.

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  1. A Aints

    Present address: 3Present address: Department of Medicine, Division of Hemato-Oncology, Tartu University, Tartu, Estonia.,

Authors and Affiliations

  1. Department of Medicine, Division of Hematology, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden

    A Treschow, C Unger, A Aints, U Felldin, J Aschan & M S Dilber

  2. Centre for Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Stockholm, Sweden

    J Aschan

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Correspondence to M S Dilber.

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Treschow, A., Unger, C., Aints, A. et al. OuaSelect, a novel ouabain-resistant human marker gene that allows efficient cell selection within 48 h. Gene Ther 14, 1564–1572 (2007). https://doi.org/10.1038/sj.gt.3303015

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