A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans

Duverger, Yohann; Belougne, Jérôme; Scaglione, Sarah; Brandli, Dominique; Béclin, Christophe; Ewbank, Jonathan J.

doi:10.1093/nar/gkl1046

Cited by 28 publications

(37 citation statements)

References 24 publications

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“…We previously reported an adjustment of the Biosort that allowed one 96-well plate to be analyzed in 36 minutes 19 . The machine's manufacturer UBI now offers a modification of the Biosort to decrease further the analysis time.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Automated High-throughput Genome-wide RNAi Screens in <em>C. elegans</em>

Squiban

Belougne

Ewbank

et al. 2012

JoVE

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RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing 9 . When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

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Section: Discussionmentioning

confidence: 99%

Quantitative and Automated High-throughput Genome-wide RNAi Screens in <em>C. elegans</em>

Squiban

Belougne

Ewbank

et al. 2012

JoVE

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“…Through such detailed investigation, we sought to optimize Mos1 tools and reagents for highthroughput screenings. Through a thorough analysis of multiple parameters (cultivation and heat-shock conditions, nematode sorter transfer protocols, PCR detection protocols) and after a number of further modifications that were made to the initial protocol (generation and frequent recrosses of new double transgenic strains in which the transposon and transposase extrachromosomal arrays were associated with fluorescent reporters of different colours), the necessary automation of the process was successfully put in place (Duverger et al 2007;Granger et al 2004). …”

Section: Workpackage 1: Optimization/automation Of Mos1 Transposon-bamentioning

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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“…In addition, within the frame of the NemaGENETAG project (see http://elegans.imbb. forth.gr/nemagenetag/ and article by Daphne Bazopoulou and Nektarios Tavernarakis in this issue), the team of Jonathan Ewbank (CIML, Marseille, France) has developed a semi-automated high-throughput approach they used to generate and recover 55,000 clonal C. elegans strains all carrying at least 1 Mos1 insertion (Duverger et al 2007). Part of this collection was analyzed by inverse PCR and about 13,000 Mos1 insertions were mapped.…”

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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A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans

Cited by 28 publications

References 24 publications

Quantitative and Automated High-throughput Genome-wide RNAi Screens in <em>C. elegans</em>

Quantitative and Automated High-throughput Genome-wide RNAi Screens in <em>C. elegans</em>

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

Manipulating the Caenorhabditis elegans genome using mariner transposons

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