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Engineering eukaryotic signal transduction with RNAi: Enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1
Author(s) -
March John C.,
Bentley William E.
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20951
Subject(s) - rna interference , microbiology and biotechnology , biology , gene silencing , rna silencing , signal transduction , cell growth , cell cycle , cell culture , tor signaling , cell , rna , gene , biochemistry , genetics
RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double‐stranded RNA (dsRNA) for the post‐transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin‐mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro‐synthesized dsRNA, we have created a tunable and specific metabolic “throttle” that, like insulin, apparently increases the specific growth rate of S2 cells in a dose‐dependent manner. This “throttle,” augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 µg/mL dsTSC1 and by over 20% by the addition of 30 µg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have “tuned” a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated. © 2006 Wiley Periodicals, Inc.