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.
Keywords: AMP, antimicrobial peptide/protein; BR021, Harmonia axyridis antimicrobial peptide BR021; BSA, bovine serum albumin; D. melanogaster S2 cells; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; GMP, good manufacturing practice; GmGlv, Galleria mellonella antimicrobial peptide Gloverin; Insect cell culture; Monoclonal cell line; OD600, optical density at 600nm; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; RMCE, recombinase mediated cassette exchange; Recombinant protein expression; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; SFM, serum free medium; Sf9, clonal isolate of Spodoptera frugiperda Sf21 cells; Single-cell cloning; Stably transformed; rS2, recombinant Drosophila melanogaster Schneider 2 cells.