A Tightly Regulated Inducible Expression System for Conditional Gene Knock-Outs and Dominant-Negative Genetics in Trypanosoma Brucei

Mol Biochem Parasitol. 1999 Mar 15;99(1):89-101. doi: 10.1016/s0166-6851(99)00002-x.

Abstract

First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Gene Deletion
  • Gene Expression Regulation*
  • Genes, Reporter
  • Genetic Markers
  • Genetic Vectors / genetics*
  • Luciferases / biosynthesis
  • Luciferases / genetics
  • Promoter Regions, Genetic
  • Repressor Proteins / genetics
  • Tetracycline / pharmacology
  • Transformation, Genetic
  • Trypanosoma brucei brucei / genetics*
  • Trypanosoma brucei brucei / growth & development
  • Trypanosoma brucei brucei / metabolism*
  • Viral Proteins

Substances

  • Genetic Markers
  • Repressor Proteins
  • Viral Proteins
  • tetracycline resistance-encoding transposon repressor protein
  • Luciferases
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Tetracycline