Comparative analysis of inducible expression systems in transient transfection studies

Anal Biochem. 2004 Nov 1;334(1):9-19. doi: 10.1016/j.ab.2004.07.011.

Abstract

Ectopic protein expression in mammalian cells is a valuable tool to analyze protein functions. Increasingly, inducible promoters are being used for regulated gene expression. Here, we compare expression maxima, induction rates, and "leakiness" of the following promoter systems: (I) two tetracycline-responsive Tet systems (Tet-On, Tet-Off), (II) the glucocorticoid-responsive mouse mammary tumor virus promoter (MMTVprom), (III) the ecdysone-inducible promoter (EcP), and (IV) the T7 promoter/T7 RNA polymerase system (T7P). The systems were analyzed by expressing an enhanced green fluorescent protein (EGFP) luciferase fusion reporter protein in transiently transfected cells. Expression was assessed qualitatively by fluorescence microscopy of the EGFP component and quantitatively by measuring the enzymatic activity of the luciferase component of the fusion protein. Basal expression levels ("leakiness") were ranked Tet-On>Tet-Off>MMTVprom>EcP>T7P. Induction rates were EcP>MMTVprom>T7P>Tet-Off>Tet-On. Expression maxima were ranked. Tet-On>Tet-Off>MMTVprom>EcP>T7P. To increase T7-promoter-mediated expression we inserted an internal ribosomal entry site element into the T7 expression cassette. In presence of T7 RNA polymerase this modified T7 promoter achieved expression levels of 42% of a Rous Sarcoma virus promoter, while keeping basal expression extremely low.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacteriophage T7 / genetics
  • Cell Line
  • DNA-Directed RNA Polymerases / genetics
  • Dexamethasone / pharmacology
  • Doxycycline / pharmacology
  • Ecdysone / genetics
  • Ecdysterone / analogs & derivatives*
  • Ecdysterone / pharmacology
  • Gene Expression Regulation, Viral / drug effects*
  • Humans
  • Mammary Tumor Virus, Mouse / genetics
  • Promoter Regions, Genetic / drug effects
  • Promoter Regions, Genetic / genetics*
  • Transfection / methods*
  • Transfection / standards
  • Viral Proteins

Substances

  • Viral Proteins
  • Ecdysone
  • Ecdysterone
  • Dexamethasone
  • ponasterone A
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Doxycycline