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ACS Chem Biol,
2006]
Identification of bioactive molecules and their targets impedes the process of drug development. In a recent paper, a genetically tractable organism, the Caenorhabditis elegans worm, is shown to be a viable screening system in which the drug target and the pathway it activates can be readily identified.
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Neuromuscul Disord,
2004]
In her commentary on our recently published paper, A. de Luca questions the approach consisting in screening random molecules on a dystrophin-deficient invertebrate model (C. elegans) in order to identify potential therapeutic clues.
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ACS Chem Biol,
2007]
Invertebrate animal models (mainly the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster) are gaining momentum as screening tools in drug discovery. These organisms combine genetic amenability, low cost, and culture conditions compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a physiological context. On the down side, protein divergence between invertebrates and humans causes a high rate of false negatives. Despite important limitations, invertebrate models are an imperfect yet much needed tool to bridge the gap between traditional in vitro and preclinical animal assays.
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Neuromuscul Disord,
2002]
We investigated the function of dystrophin in the nematode Caenorhabditis elegans. Although nematodes and mammals diverged early in evolution, their muscles share many structural and molecular features, thus rendering C. elegans relevant as a model to study muscle function. Dystrophin, dystrobrevin, dystroglycans and several sarcoglycans have conserved homologues in the genome of C. elegans. The major strength of the model comes from its genetic tractability, which allows the quick and easy manipulation of gene expression, either to inactivate genes, or to create transgenic animals. Over the last 2 years, work on C. elegans dystrophin has led to the identification of a putative new member of the dystrophin-glycoprotein complex, and has brought additional data suggesting that dystrophin mutations affect ion
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CRC Methods in Cellular and Molecular Neuropathology Series,
1999]
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Med Sci (Paris),
2003]
C. elegans as a model for human inherited degenerative diseases. The nematode C. elegans is an established model for developmental biology. Since the early 90's, this simple model organism has been increasingly used for studying human disease pathogenesis. C. elegans models based either on the mutagenesis of human disease genes conserved in this nematode or transgenesis with disease genes not conserved in C. elegans show several features that are observed in mammalian models. These observations suggest that the genetic dissection and pharmacological manipulation of disease-like phenotypes in C. elegans will shed light on the cellular mechanisms that are altered in human diseases, and the compounds that may be used as drugs. This review illustrates these aspects by commenting on two inherited degenerative diseases, Duchenne's muscular dystrophy and Huntington's neurodegenerative disease.
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International C. elegans Meeting,
1999]
With the genome sequence being complete, it is essential to determine the function of each predicted gene. To facilitate the cloning of genes, it is also important to create as many polymorphisms as possible. With these goals in mind, we have launched a large scale project whose long-term objective is to isolate insertions of transposable elements in most C. elegans genes. To do so, we have chosen to use techniques based on the random insertion of natural worm transposons, an approach which has been pioneered in the Plasterk lab. Starting from mutator backgrounds, we generate clones and determine the position of new insertions by a modification of the transposon insertion display protocol described in the WBG (Vol 14, ndeg4 page 20), in which DNA flanking transposons can be amplified by anchored PCR, and sequenced. Assuming ideal statistical conditions (non-biased insertion sites, independence of insertion events and no intergenic regions), the Poisson distribution would predict that 30,000 independent sequence reads would be enough to hit 80% of the estimated 19,000 C. elegans at least once. In practice, since the last of these three conditions cannot be met (and assuming the other two are), 30,000 insertions should lead to an insertion every few kilobases, which would give a polymorphism coverage of the genome much higher than the current one. It is expected that approximately a quarter of these insertions will be in coding sequences and UTRs (representing. potential mutations). This project is a complementary alternative to the gene-directed PCR-based search for deletions. We also believe that this project (currently estimated at $2-4 Million for 30,000 sequence reads) is competitive for cost and labor compared to gene targeting. Furthermore, transposon insertions should potentially provide a wider spectrum of alterations, which will be needed as genetic tools besides the knock-outs. We are currently running small-scale pilot experiments on a few hundred clones to optimize the protocols and validate the approach.
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European Worm Meeting,
2006]
Vallin E., Granger L., Martin E. & Segalat L.. A collection of Caenorhabditis elegans insertion mutants is being created as part of the NemaGENETAG project. The aim is to produce characterized transposon-tagged mutants for the European and international scientific community. We use the Drosophila mariner element Mos-1, which has been shown to be active in C.elegans. The J. Ewbank laboratory (Marseille) generates and provides us with Mos-positive strains, which we molecular characterize. The localization of the insertion is determined by inverse PCR and sequencing. The sequence is compared with the C. elegans genome sequence to determine the insertion sites using the blast program. At the beginning of 2006, we have characterized approximately 2000 strains. Strains are currently analyzed sequentially with enzymes Hae III and Mbo I. Approximately 70% of the strains received from Marseille give a workable PCR band in these conditions. The percentage of PCR bands that give an unambiguous genome localization is approximately 66%. These strains are frozen in triplicate in both -80C freezer and liquid nitrogen. In case of request or for quality control, strains are thawed and PCR-tested with a Mos primer and an insertion-specific primer to check the presence on the insertion. On 30 strains tested in recovery tests, 28 were positive. These results prove that the insertions are stable and can be easily recovered from frozen samples. The aim of the project is to generate and provide strains for the worm community. The list of the insertions can be found on a database available on the web.
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Br J Pharmacol,
2010]
Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.
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European Worm Meeting,
2006]
During the evolution, nematodes and mammals diverged but nematodes muscles preserved many structural and molecular features similar to mammal muscles. Therefore C. elegans is an excellent model to study muscle function. Since 2005, our laboratory is part of MYORES European research network. This project, financed for 5 years by the European Commission, focuses on basic issues of muscle physiology and eventually the development of therapies for muscle pathologies. MYORES aggregates 6 technical platforms, which utilize different model organisms (Nematodes, Drosophila, Zebra fish, chick and mouse) to accelerate studies on normal and aberrant muscle development. All data about muscular development assembled by MYORES will be integrated into the MYORES database: Myobase. Our laboratory is in charge of the C. elegans RNAi platform. We use RNA interference in C. elegans to study normal and pathological functions of muscle specific proteins. The purpose of this platform within MYORES is to give access to C. elegans to researchers familiar with other models. In collaborative projects, we are currently doing gene inactivation experiments on MyoD, Pax-3 and Six genes, which are important developmental switches for the development of vertebrate muscles. We will present data obtained in this work.