Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elem ents are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator prote in (tTA(s)*). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter cont ains a tetracycline-responsive element, which is the binding site for tTA(s )*. Tetracycline prevents tTA(s)* from binding to the tetracycline-responsi ve element, rendering the promoter virtually silent. In the absence of tetr acycline, tTA(s)* binds to its target sequence and strongly induces gene ex pression. The kinetics of activation and repression of the system were moni tored using luciferase as a reporter. The results reveal efficient inhibiti on of gene expression by low concentrations of tetracycline and an inductio n of gene expression by several orders of magnitude within a few hours afte r removal of tetracycline. Green fluorescent protein (GFP) provided informa tion about the effects of modulation of the tetracycline concentration on g ene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population e xpress the reporter gene. (C) 2000 Elsevier Science B.V. All rights reserve d.