{Reference Type}: Journal Article
{Title}: Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression.
{Author}: Zitzmann J;Schreiber C;Eichmann J;Bilz RO;Salzig D;Weidner T;Czermak P;
{Journal}: Biotechnol Rep (Amst)
{Volume}: 19
{Issue}: 0
{Year}: Sep 2018
暂无{DOI}: 10.1016/j.btre.2018.e00272
{Abstract}: 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.