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Brielmeier, M. ; Bechet, J.-M.* ; Falk, M.H.* ; Pawlita, M.* ; Polack, A. ; Bornkamm, G.W.

Improving stable transfection efficiency : antioxidants dramatically improve the outgrowth of clones under dominant marker selection.

Nucleic Acids Res. 26, 2082-2085 (1998)
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Many cell lines are sensitive to growth at low cell density and undergo apoptosis induced by oxidative stress if the cell density is decreased below a critical threshold. In stable transfection experiments this cell density-dependent growth may be the limiting factor, since during drug selection the cell density falls below the critical threshold, precluding outgrowth of transfected clones. We describe here a simple protocol for the establishment of stably transfected human B cell lines making use of the protective action of antioxidants. The protocol includes: (i) seeding the cells in medium supplemented with sodium pyruvate, alpha-thioglycerol and bathocuproine disulfonate; (ii) delaying the onset of dominant marker selection to improve recovery of the cells after electroporation. Stably transfected clones have thus been obtained from Burkitt's lymphoma lines, which have been regarded as untransfectable. Using this protocol the stable transfection efficiency with episomal plasmids approaches the transient transfection efficiency, indicating that virtually every transfected cell can be established as a stably transfected clone. This protocol should also prove useful for other cell lines, e.g. neuronal cells, having similar sensitivities to oxidative stress.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords VIRUS NUCLEAR ANTIGEN-2; MAMMALIAN-CELLS; OXIDATIVE STRESS; LYMPHOMA CELLS; CYTO-TOXICITY; B-CELLS; ELECTROPORATION; EXPRESSION; GENE; DNA
ISSN (print) / ISBN 0305-1048
e-ISSN 1362-4962
Journal Nucleic Acids Research
Quellenangaben Volume: 26, Issue: 9, Pages: 2082-2085 Article Number: , Supplement: ,
Publisher Oxford University Press
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Clinical Molecular Biology and Tumor Genetics (K.MOLBI)