A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei

First-generation inducible expression vectors for Trypanosoma brucei utiliz ed a single tetracycline-responsive promoter to drive expression of an expe rimental gene, in tandem with a drug-resistance marker gene to select for i ntegration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activit y of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approac h, for expressing highly toxic products and generating conditional gene kno ck-outs, using back-to-back constitutive T7 and tetracycline-responsive PAR P promoters to drive expression of the selectable marker and test gene, res pectively. Transformants are readily obtained with these vectors in the abs ence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-e xpressing T7 RNA polymerase and Tet repressor, and consistently show tetrac ycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced ba ckground expression of a luciferase reporter averages no more than one mole cule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the produ ction of functional gene knock-outs through regulated expression of an expe rimental gene in a null-mutant background. (C) 1999 Elsevier Science B.V. A ll rights reserved.