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We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Original publication

DOI

10.1038/74493

Type

Journal article

Journal

Nat Biotechnol

Publication Date

04/2000

Volume

18

Pages

429 - 432

Keywords

Alphavirus, Animals, CHO Cells, Cell Line, Cricetinae, Feedback, Gene Expression Regulation, Viral, Kinetics, RNA Replicase, Recombinant Fusion Proteins, Replicon, Temperature, Transfection