Project objectives

We plan to generate a comprehensive collection of ordered transposon tagged C. elegans alleles. In addition, we will develop alternative systems based on the well characterized Minos transposon, both as contingency measures to address potential bias and efficiency issues, but also to extend functionality by allowing the construction of vehicles to integrate exogenous DNA into the genome, and the development of gene-trapping methodologies.

Our initiative has 3 clear objectives, exemplified in the form of 5 distinct workpackages with measurable and verifiable endpoints and deliverables. Specifically:

Objective 1. Optimization/automation of the Mos1-based system for large-scale mutagenesis

The Mos1 system has already been established as an efficient tool for gene-tagging in C. elegans. It is based on the generation of transgenic worm strains carrying two independent extrachromosal arrays, one that encodes the Mos1 transposase under the control of an inducible promoter, the other that includes copies of the Mos1 transposon. Induction of transposase expression leads to the mobilisation of the transposon and its integration within the C. elegans genome. The extrachromosal arrays are not inherited in a Mendelian fashion and can be easily lost in subsequent generations, resulting in the stabilisation of the Mos1 insertion. We will further characterize this system in terms of insertion bias and mutagenicity. Through such detailed characterization, we will seek to optimize Mos1 tools and reagents for high-throughput screenings. Scaling up transposon-mediated gene-tagging to the whole-genome level requires considerable investment in the development of technology platforms that will allow automation and streamlining of various processes. Our participant, MAIA Scientific (Participant #4), will contribute towards this goal by deployment/development of the required solutions in close collaboration with the participants involved in generating and evaluating large collections of transposon insertion mutants.

Objective 2. Development of novel transposon-based systems for mutagenesis, transgenesis and genome engineering in C. elegans

Development of other transposons systems is important for two reasons. First, all transposons have preferential insertion sites in genomes. We predict that even after isolating a very large number of Mos1 insertions, specific regions of the genome will be under-sampled. Another transposon would have a distinct insertion bias and would provide a way to target the genes that are found in such regions. Second, transposons can used to introduce foreign sequences into the host genome. This feature is widely used for enhancer-trap systems or tissue-specific expression systems. Mos1 can accommodate exogenous DNA but the frequency of transposition decreases exponentially with the size of the insert, such that a maximum of only 300 bp of exogenous DNA can be included in recombinant Mos1 transposons . To circumvent these issues we plan to develop alternative transposon systems in C. elegans based on the well-characterised and widely used Minos transposable element.

Localized transposon insertions also represent an entry point to further manipulate the locus where they inserted. First, imprecise excision of mariner transposons causes various types of gene lesions such as gene deletions. Mos1 re-mobilization followed by imprecise repair can be achieved in C. elegans. By reintroducing a Mos1 transposase expression transgene, it is possible to identify excision events that cause deletions or small insertion footprints in the gene that is tagged with Mos1 insertion. Ongoing experiments indicate that Mos1 re-excision is very efficient: using the mutant unc-5(ox171::Mos1) which contains a Mos1 element inserted into the seventh exon of the unc-5 gene, we have demonstrated that greater than 6.3 % of the chromosomes experience excision after heat-shock induction of Mos1 transposase expression. We could also demonstrate that after excision, the double-strand break was repaired from the homologous chromosome. This repair mechanism will regenerate a Mos1 copy at the site of excision. However, repair is inhibited when chromosome pairing is disrupted. These features of Mos1 re-excision will be used to recover imprecise excision events at high frequency. This tool combined with a comprehensive library of Mos1 insertions would provide a general resource to knock-out most of the C. elegans genes at low cost. Second, gene conversion following transposon excision has been exploited to copy information into a genome in a site-specific manner. In Drosophila, Glooret al. (Science, 1991, 253, 1110) were able to copy sequences from one chromosomal locus into another. In C. elegans, Plasterk et al.(EMBO J., 1992, 11, 287) were able to copy homologous sequences from an extrachromosomal array into the genome at the native chromosomal locus. However, Tc1 induced transgene-instructed gene conversion was rare, occurring at a frequency of 2X10^-5, preventing this technique from being widely utilized in C. elegans research. Mos1 provides an alternative to circumvent this limitation. First, Mos1 re-mobilization is extremely efficient. Second, using balancer chromosomes, it is possible to inhibit gene conversion from the homologous chromosome. Such events would otherwise out-compete recombination with the extrachromosomal array that will be used as a template for double-strand break repair. If exogenous sequences could be introduced efficiently into the C. elegans genome via Mos1-mediated transgene-instructed gene conversion, a library of strains containing Mos1 insertions in every gene would provide a feasible alternative to homologous recombination techniques that are not available in C. elegans.

Objective 3. Construction of an ordered library of transposon-tagged alleles covering at least 85% of the C. elegans gene complement.

Our aim is to use the tools and technologies described above to generate a comprehensive collection of transposon-tagged nematode genes. Such a mutant collection will provide an extremely valuable resource because it will accelerate our understanding of gene function, which is a major challenge in biology. Since, approximately 50% of human genes have a C. elegans homologue and more than 65% of human disease genes are represented in the nematode genome, a library of transposon-tagged genes will provide ready-made models and reagents to tackle human pathologies. We intend to organize the necessary infrastructure necessary for maintaining and freely distributing this resource to interested colleagues all over the world. C. elegans strain stocks can be kept frozen and thawed easily, which greatly facilitates the conservation and distribution of mutant collections.