An extrachromosomal, inducible expression system for Dictyostelium discoideum

Abstract

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.

Introduction

Ectopical gene expression is an indispensable tool for the analysis of gene function. High concentrations of gene product can be toxic to cells, which necessitates control over the level of gene expression. Several inducible expression systems have been developed for this purpose. In Dictyostelium, inducible transcriptional regulation has been described using a folate-dependent promoter element (Blusch et al., 1992) and using a DNA-damage induced promoter (Gaudet et al., 2001). A translational control system has also been developed, where a suppressor tRNA gene allows translation through a UAG stop codon (Dingermann et al., 1992, Pang et al., 2001). The system that is currently most widely used makes use of a recombinant transcription factor that activates transcription in a tetracycline-dependent manner (Gossen and Bujard, 1992). The reason for its popularity is the high specificity and affinity by which the transactivator binds to the inducible promoter, resulting in a tight control of gene transcription. The levels of basal and induced expression of cells that are transfected with a tetracycline-inducible vector differs from clone to clone and must determined individually. In the most favorable clones, expression of a reporter gene can be induced over a 100,000-fold range (Gossen and Bujard, 1992). The tetracycline-responsive system was originally developed for mammalian cells, but has been adapted for use in Dictyostelium (Blaauw et al., 2000). In this system, the transactivator is first integrated into the genome. Obtained stable cell lines are then transfected with an extrachromosomal vector containing the controlled gene of interest. As in the mammalian system, the inducibility of gene transcription depends on the site and copy number of the integrated transactivator and expression levels need to be determined individually for each clone.

We have described a new set of modular expression vectors where transcription of the gene of interest is under the control of the act15 promoter (Veltman et al. this issue), which results in high levels of transcription in both vegetative and developed cells. In this paper, we put all components of the existing 2-plasmid tetracycline-inducible system onto a single, Ddp1-based extrachromosomal vector. Despite the insertion of the transactivator gene, the size of the inducible vectors is still smaller than that of other Ddp1-based vectors such as pLittle and pJK1, that are used for constitutive expression (Levi et al., 2000, Pitt et al., 1992). Most importantly, since all components reside on one extrachromosomal vector, highly reproducible results are obtained from independent transfections without the need for clonal selection.