With their first inducible RNA-based system for gene activation in the roundworm Caenorhabditis elegans (C. elegans), two researchers from the University of Constance have filled a significant gap in the research and use of genetic switches. The new approach was developed in the context of a joint research project led by dr. Martin Gamerdinger (Department of Biology) and Professor Jörg Hartig (Department of Chemistry) at the Collaborative Research Center of the University of Constance 969 "Chemical and biological principles of cellular proteostasis," which is funded by the German Research Foundation (DFG ). Sharing their respective skills in the area of C. elegans and the development of RNA-based genetic switches, the researchers were able – for the first time – to successfully induce a gene in the animal model using an RNA-based genetic switch. They have also been able to establish a new model of disease inducing for Huntington's disease that opens up new opportunities for research and application.
Their card "A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans"is published on 30.01.2019 in the online journal Communications of nature.
The nematode C. elegans it is widely used for research in cell biology and development and as a model system for the study of human diseases. This animal model of research enjoys particular popularity because its anatomy is extremely simple, since its transparency allows live-cell microscopy and because many of its genes are preserved in the evolution in man. "However, what I found rather frustrating is that we did not have an inducible system for this animal model that would allow us to activate genes at will," explains Dr. Martin Gamerdinger, initiator of research collaboration and project leader in CRC 969 "Chemistry and biological principles of cellular proteostasis" at the University of Constance. As a result of the close collaboration with the Chemistry and Synthetic Biology group of the Nucleic Acids led by Professor Jörg Hartig, Gamerdinger and his colleagues have been able to establish a particularly convenient and efficient system for inducing a gene in the C. elegans animal model.
Professor Hartig's team, which also includes Dr. Lena Wurmthaler, the first author of the work, investigates unusual structures and characteristics of nucleic acids, in particular catalytically active ribozymes and the so-called riboswitch, which can be used to activate or deactivate single genes. A main purpose of RNA (ribonucleic acid) in biological cells is to translate genetic information into proteins; as mRNA serves as an information carrier. The introduction of autonomous cleavage ribozymes towards mRNA molecules leads to the decay of mRNA and, ultimately, to the deactivation of the gene. However, the activity of ribozyme – and therefore gene expression – can be controlled by ligand-dependent ribozymes. The inducible system of researchers is based on the lecand tetracycline, which belongs to the group of antibiotics. Tetracycline binds to the RNA molecule via a so-called RNA aptamer and inhibits the activity of ribozyme. This, in turn, stabilizes the mRNA, which is then translated into proteins. As a result, the desired gene can be turned on. "The fantastic thing about the new genetic switch is that it can be used at all stages of development C. elegans, ie through all the larval stages and in the adult animal, which until now was not possible ", explains Dr. Lena Wurmthaler, among the main advantages of this new approach is that no additional proteins are required Regulators to regulate gene expression: all that is necessary to ignite the gene is necessary to insert the ribozyme together with the aptamer (aptazyme or ribozyme dependent on the ligand) in the mRNA and C. elegans tetracycline. Lena Wurmthaler: "We need very little space for coding and no additional protein factor expressed, in a single step, we can convert any gene of interest into a gene inducible to tetraiclocin."
Supported by PhD researchers Monika Sack and Karina Gense, Martin Gamerdinger and Lena Wurmthaler were able to test their new system by changing the expression of the red fluorescent protein mCherry in various cell types. C. elegans. "Having such a simple and highly effective inducible system is a big step forward for the whole C. elegans field, "concludes Gamerdinger, the two researchers expect their new system to be relevant in a number of contexts, especially in medical research, successfully activating a mutant gene that encodes the Huntingtin (Htt) protein and causes Huntington's disease. In humans, neurodegenerative brain disease, they were able to establish and study a new model of inducible disease as part of their study.
Unlike a healthy gene, the variant of the Huntingtin gene used in the study encodes an abnormally extensive polyglutamine sequence. For their experiment, the two researchers created a particularly aggressive disease model with 109 sequential polyglutamines (Htt109Q). In a demonstrable way, the feeding of the tetracycline of worms has led to an increase in the aggregation of Huntingtin in C. elegans, which caused paralysis and, when targeted specifically to neurons, severe loss of motor coordination, which documents the neurotoxicity of the induced gene. Using this approach, Martin Gamerdinger and Lena Wurmthaler were able to establish the first inducible model of Huntington's disease in C. elegans. Since it is now possible to induce toxic proteins such as huntingtin at will, it will be possible in the future to generate and study additional disease models based on extremely toxic proteins.
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