Abstract
We have demonstrated the RNA interference-based interruption of cellular controllers to increase recombinant protein yield in Drosophila Schneider 2 (S2) cell culture. Double-stranded RNA (dsRNA) was enzymatically synthesized in vitro and transfected into stable cell lines expressing green fluorescent protein (GFP) under an inducible promoter. Components of cell cycling (CycE and ago) were silenced with dsRNA homologous to a 700-nucleotide section of their respective mRNA transcripts. Silencing ago and CycE resulted in increases in product yield of up to 1.8-fold and 4-fold, respectively, relative to a control transfected with nuclease-free water. It is surprising to note that nearly complete silencing of CycE resulted in no significant change in GFP fluorescence after 24 h, and a decrease in fluorescence after 72 h. By partially silencing CycE, however, we were able to retain 80% of the cells in G1 (48-h sample) and increase GFP synthesis by fourfold. Implications for protein synthesis processing are discussed.
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Acknowledgements
The authors express gratitude to Dr. David Mosser at the University of Maryland for help with flow cytometry. This work was funded by NIH grant 1R01GM70851-01.
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March, J.C., Bentley, W.E. RNAi-based tuning of cell cycling in Drosophila S2 cells—effects on recombinant protein yield. Appl Microbiol Biotechnol 73, 1128–1135 (2007). https://doi.org/10.1007/s00253-006-0560-x
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DOI: https://doi.org/10.1007/s00253-006-0560-x