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 elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTA(s)(*)). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.
Blaauw
M
M
Cell Engineering Facility GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. m.blaauw@chem.rug.nl
Linskens
M H
MH
van Haastert
P J
PJ
D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000187:drug effects* / Q000235:genetics
D000199:Actins; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline