Abstract
The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3’-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington’s disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.
Highlights
The nematode Caenorhabditis elegans represents an important research model
The utilization of ribozyme switches for regulating gene expression in C. elegans has several distinct advantages compared to the existing methods
Ribozyme-based switches are powerful research tools to achieve conditional gene expression in model organisms like bacteria, Saccharomyces cerevisiae or human cell culture[6]. To test whether such systems are adaptable for the animal model C. elegans, we utilized a type III hammerhead ribozyme (HHR) from Schistosoma mansoni[9] inserted into the 3’-UTR of a pharynx-specific mCherry reporter gene
Summary
The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Major drawbacks of this method are the use of heat stress as the inducing trigger that can have an impact on cell physiology and gene function, and the non-reversible switching mode that occurs on the DNA level Another method employs a transcription-based repressible binary expression system originating from the mold fungus Neurospora crassa[3,4]. Apart from using condition-responsive or effector-responsive transcription factors, small self-cleaving ribozymes have been utilized for controlling gene expression on a post-transcriptional level For this purpose, ribozymes are inserted into non-coding regions of the mRNA of interest. The post-transcriptional regulation mechanism is part of the mRNA and intrinsically allows for robust expression control It needs comparably little coding space and can be introduced into untranslated regions of mRNAs. Further, the gene of interest can be expressed from natural promoters preserving the tissue-specific expression pattern, which is highly relevant if functional studies are carried out. Using the ribozymebased switch we established inducible C. elegans polyglutamine (polyQ) Huntington’s disease models
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