%0 Journal Article
%T Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression.
%A Zitzmann J
%A Schreiber C
%A Eichmann J
%A Bilz RO
%A Salzig D
%A Weidner T
%A Czermak P
%J Biotechnol Rep (Amst)
%V 19
%N 0
%D Sep 2018
%M 29998071
暂无%R 10.1016/j.btre.2018.e00272
%X The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without using fetal bovine serum and/or feeder cells pretreated by irradiation or exposure to mitomycin. Highly productive clones of Drosophila melanogaster S2 cells were prepared in a two-step procedure, comprising the establishment of a polyclonal population and subsequent single cell isolation by limiting dilution. Necessary growth factors were provided by co-cultivation of single transformants with untransfected feeder cells, which were later removed by antibiotic selection. Enhanced expression of EGFP and two target peptides was confirmed by flow cytometry and dot/western blotting. Highly productive clones were stable, showed a uniform expression profile and typically a sixfold to tenfold increase in cell-specific productivity.