Brielmeier M, Béchet J M, Falk M H, Pawlita M, Polack A, Bornkamm G W
Institute for Clinical Molecular Biology and Tumor Genetics, GSF, National Research Institute for Environment and Health, Marchioninistrasse 25, D-81377 München, Germany.
Nucleic Acids Res. 1998 May 1;26(9):2082-5. doi: 10.1093/nar/26.9.2082.
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.
许多细胞系在低细胞密度下对生长敏感，如果细胞密度降至临界阈值以下，会因氧化应激而发生凋亡。在稳定转染实验中，这种细胞密度依赖性生长可能是限制因素，因为在药物筛选过程中，细胞密度会降至临界阈值以下，从而阻止转染克隆的生长。我们在此描述一种利用抗氧化剂的保护作用建立稳定转染的人B细胞系的简单方案。该方案包括：（i）将细胞接种在补充有丙酮酸钠、α-硫甘油和二磺酸 bathocuproine 的培养基中；（ii）延迟显性标记选择的开始时间，以提高电穿孔后细胞的回收率。因此，已从被认为不可转染的伯基特淋巴瘤细胞系中获得了稳定转染的克隆。使用该方案，游离质粒的稳定转染效率接近瞬时转染效率，这表明几乎每个转染细胞都可以建立为稳定转染的克隆。该方案对于对氧化应激具有相似敏感性的其他细胞系（例如神经元细胞）也应该是有用的。
