A temperature-regulated replicon-based DNA expression system

Nat Biotechnol. 2000 Apr;18(4):429-32. doi: 10.1038/74493.

Abstract

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.

MeSH terms

  • Alphavirus / genetics*
  • Animals
  • CHO Cells
  • Cell Line
  • Cricetinae
  • Feedback
  • Gene Expression Regulation, Viral*
  • Kinetics
  • RNA Replicase / genetics
  • RNA Replicase / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Replicon / genetics*
  • Temperature
  • Transfection / methods

Substances

  • Recombinant Fusion Proteins
  • RNA Replicase