Characterization of Mos1-Mediated Mutagenesis in Caenorhabditis elegans

Williams, Daniel C.; Boulin, Thomas; Ruaud, Anne Françoise; Jørgensen, Erik M.; Bessereau, Jean-Louis

doi:10.1534/genetics.104.038265

Cited by 43 publications

(46 citation statements)

References 23 publications

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“…Second, MOS1 is not sufficiently effective to promote a large-scale detectable level of transposition. Results obtained by Williams et al (2005), and Robert and Bessereau (2007) using Mos1 in C. elegans do not support this hypothesis. Third, MOS1 might be down-regulated by post-translational modification, as suggested by the present data.…”

Section: Discussionmentioning

confidence: 85%

Mariner Mos1 transposase optimization by rational mutagenesis

et al. 2009

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Mariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity.

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Section: Discussionmentioning

confidence: 85%

Mariner Mos1 transposase optimization by rational mutagenesis

et al. 2009

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“…Parameters for optimal Mos1 transposition were characterized to optimize the efficiency of Mos1-mediated insertional mutagenesis (Williams et al 2005). Mos1 inserts throughout the C. elegans genome into TA dinucleotides with a slight preference for introns (Granger et al 2004).…”

Section: Custom Knockout and Knock In Alleles By Transgene-instructedmentioning

confidence: 99%

“…When mutagenesis is performed under optimal conditions, Mos1 is only 10 times less mutagenic than the chemical agent EMS (for a detailed protocol see Bessereau 2006a;Boulin and Bessereau 2007). On average, 2.5 Mos1 insertions are recovered per animal (Williams et al 2005). Because of this low copy number of Mos1 insertions per mutant, the mutagenic insertion responsible for a mutant phenotype can be quickly separated from unlinked insertions by out-cross and mapped using inverse PCR.…”

Section: Custom Knockout and Knock In Alleles By Transgene-instructedmentioning

confidence: 99%

Manipulating the Caenorhabditis elegans genome using mariner transposons

Robert

Bessereau

2009

Genetica

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Tc1, one of the founding members of the Tc1/mariner transposon superfamily, was identified in the nematode Caenorhabditis elegans more than 25 years ago. Over the years, Tc1 and other endogenous mariner transposons became valuable tools for mutagenesis and targeted gene inactivation in C. elegans. However, transposition is naturally repressed in the C. elegans germline by an RNAi-like mechanism, necessitating the use of mutant strains in which transposition was globally derepressed, which causes drawbacks such as uncontrolled proliferation of the transposons in the genome and accumulation of background mutations. The more recent mobilization of the Drosophila mariner transposon Mos1 in the C. elegans germline circumvented the problems inherent to endogenous transposons. Mos1 transposition strictly depends on the expression of the Mos transposase, which can be controlled in the germline using inducible promoters. First, Mos1 can be used for insertional mutagenesis. The mobilization of Mos1 copies present on an extrachromosomal array results in the generation of a small number of Mos1 genomic insertions that can be rapidly cloned by inverse PCR. Second, Mos1 insertions can be used for genome engineering. Triggering the excision of a genomic Mos1 insertion causes a chromosomal break, which can be repaired by transgene-instructed gene conversion. This process is used to introduce specific changes in a given gene, such as point mutations, deletions or insertions of a tag, and to create single-copy transgenes.

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“…Mobilization of the exogenous Mos1 transposon, was accomplished in somatic cells and in the germ line of C. elegans (Bessereau et al 2001). The transformation protocol has been optimized since, allowing a significant improvement of the mutagenesis efficiency (Williams et al 2005). Presently the Mos1 system is the most widely used, fully-controllable transposable element for functional genomics purposes in C. elegans (Bessereau 2006).…”

Section: Introductionmentioning

confidence: 99%

The NemaGENETAG initiative: large scale transposon insertion gene-tagging in Caenorhabditis elegans

Bazopoulou

Tavernarakis

2009

Genetica

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The nematode Caenorhabditis elegans is a widely appreciated, powerful platform in which to study important biological mechanisms related to human health. More than 65% of human disease genes have homologues in the C. elegans genome, and essential aspects of mammalian cell biology, neurobiology and development are faithfully recapitulated in this organism. The EU-funded NemaGENETAG project was initiated with the aim to develop cutting-edge tools and resources that will facilitate modelling of human pathologies in C. elegans, and advance our understanding of animal development and physiology. The main objective of the project involves the generation and evaluation of a large collection of transposon-tagged mutants. In the process of achieving this objective the NemaGENETAG consortium also endeavours to optimize and automate existing transposon-mediated mutagenesis methodologies based on the Mos1 transposable element, in addition to developing alternatives using other transposon systems. The final product of this initiative-a comprehensive collection of transposon-tagged alleles-together with the acquisition of efficient transposon-based tools for mutagenesis and transgenesis in C. elegans, should yield a wealth of information on gene function, immediately relevant to key biological processes and to pharmaceutical research and development.

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Characterization of Mos1-Mediated Mutagenesis in Caenorhabditis elegans

Cited by 43 publications

References 23 publications

Mariner Mos1 transposase optimization by rational mutagenesis

Mariner Mos1 transposase optimization by rational mutagenesis

Manipulating the Caenorhabditis elegans genome using mariner transposons

The NemaGENETAG initiative: large scale transposon insertion gene-tagging in Caenorhabditis elegans

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