RRC ID 29023
Author Blaauw M, Linskens MH, van Haastert PJ.
Title Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells.
Journal Gene
Abstract 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.
Volume 252(1-2)
Pages 71-82
Published 2000-7-11
DOI 10.1016/s0378-1119(00)00227-4
PII S0378-1119(00)00227-4
PMID 10903439
MeSH Actins / genetics Amino Acid Sequence Animals Base Sequence DNA, Recombinant / genetics Dictyostelium / cytology Dictyostelium / genetics* Dose-Response Relationship, Drug Flow Cytometry Gene Expression Regulation / drug effects Genetic Vectors Green Fluorescent Proteins Kinetics Luciferases / drug effects Luciferases / genetics Luciferases / metabolism Luminescent Proteins / drug effects Luminescent Proteins / genetics Luminescent Proteins / metabolism Molecular Sequence Data Plasmids Promoter Regions, Genetic / genetics Recombinant Fusion Proteins / drug effects Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Response Elements / genetics Tetracycline / pharmacology* Trans-Activators / drug effects* Trans-Activators / genetics
IF 2.984
Times Cited 52
Cellular slime molds S90288 S90366