[
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.