title: RNAi-based tuning of cell cycling in Drosophila S2 cells--effects on recombinant protein yield.
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.
authors.affiliation: Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: March
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D000818:Animals; D060565:Argonaute Proteins; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D002460:Cell Line; D019927:Cyclin E / Q000037:antagonists & inhibitors; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors; D004331:Drosophila melanogaster; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000138:chemical synthesis / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
chemical_list: C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D029721:Drosophila Proteins; D012330:RNA, Double-Stranded; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins