Improving stable transfection efficiency: antioxidants dramatically improve the outgrowth of clones under dominant marker selection.
AUTOR(ES)
Brielmeier, M
RESUMO
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.
