PMID: 9547263Jun 20, 1998Paper

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

Nucleic Acids Research
M BrielmeierG Bornkamm

Abstract

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 sen...Continue Reading

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Citations

Jan 23, 2004·Journal of Immunological Methods·Masahiro KawaharaTeruyuki Nagamune
Aug 20, 2002·Journal of Hematotherapy & Stem Cell Research·Peter Buttgereit, Ingo G H Schmidt-Wolf
Aug 5, 2009·Nucleic Acids Research·Gijsbert P van NieropManuel A F V Gonçalves
Sep 9, 2005·Nucleic Acids Research·Georg W BornkammJean Feuillard
Jan 13, 2004·Genetic Vaccines and Therapy·Frank SchakowskiIngo GH Schmidt-Wolf
Dec 4, 2009·PloS One·P Thomas VernierD Peter Tieleman
Aug 26, 2014·Nature Methods·Maarten HolkersManuel A F V Gonçalves
Jan 25, 2012·Journal of Controlled Release : Official Journal of the Controlled Release Society·Nianxi ZhaoYouli Zu
Jul 1, 2008·Journal of Immunological Methods·Takahiro SogoTeruyuki Nagamune
Nov 27, 2004·Gene·Regina FeederleWolfgang Hammerschmidt
Feb 8, 2005·Vaccine·Bertrand Collet, Christopher J Secombes
Jun 10, 2016·Nucleic Acids Research·Xiaoyu ChenManuel A F V Gonçalves
Feb 14, 2018·Biotechnology Journal·Iris DelrueLia L M Verdoodt
Aug 11, 2001·Cancer Gene Therapy·P ButtgereitI G Schmidt-Wolf
Apr 25, 2008·Current Protocols in Immunology·Bernhard Fleckenstein, Armin Ensser
Nov 21, 2001·The Journal of Immunology : Official Journal of the American Association of Immunologists·M K LembergB Martoglio
Sep 25, 2017·Nature Communications·Xiaoyu ChenManuel A F V Gonçalves

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