An extrachromosomal, inducible expression system for Dictyostelium discoideum.
Inducible expression systems are essential for the expression of toxic proteins and are very convenient for proteins that induce strong side effects such as retardation of growth or development. Currently available systems for use in Dictyostelium either do not have a very tight control over expression levels or use a combination of an integrating and an extrachromosomal vector. We designed a new vector in which all components of the available 2-plasmid tetracycline-inducible system were combined onto a single extrachromosomal vector. Two types of inducible plasmids are presented, in which transcription is induced by adding or removing doxycycline, respectively. The location and orientation of the components was optimized in order to obtain a low background expression combined with high inducibility. The resulting vectors have a very low expression in the uninduced state (>1000-fold lower expression compared to that resulting from the act15 promoter), show a 10,000-fold induction of gene expression in a doxycycline concentration-dependent manner and are comparatively small (8.5 kb). With these new vectors, inducible gene expression is as easy as constitutive gene expression.
Veltman
Douwe M
DM
Cell Biochemistry, University of Groningen, Department of Biology, Haren, The Netherlands. d.veltman@beatson.gla.ac.uk
Keizer-Gunnink
Ineke
I
Haastert
Peter J M Van
PJ
D000818:Animals; D004023:Dictyostelium / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000187:drug effects; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D013752:Tetracycline / Q000494:pharmacology
D013752:Tetracycline; D004318:Doxycycline