Participant 6. Laurent Segalat (LS), PhD
CNRS-CGMC, University Claude Bernard Lyon-1, France
General: The laboratory is located on the premises of University Claude Bernard (Lyon, France) and is affiliated with both the CNRS and the University Claude Bernard. It is part of the Centre de Génétique Moléculaire et Cellulaire (CGMC), an institute of 120 people equipped with state-of-the-art equipment in molecular biology and genomics. In addition, the CGMC has tight links and shares technical resources with others laboratories of the campus federated as IFR41.
Role in the project: The laboratory will be involved in the workpackages #1, and 3.
Participation to workpackage #1: Evaluation and optimization of the current Mos1 system. Our contribution to this workpackage will initially involve examining current sets of data obtained through Mos1 pilot experiments to estimate several key parameters including 1) the statistical analysis of Mos1 insertions sites, 2) and how mutagenic insertions are. We will continue to evaluate the growing data sets obtained through Mos1 experiments to provide continuously updated estimates of these parameters. Over the whole course of the project, we will also constantly develop tools to optimize, automate, and - if possible - reduce the costs of insertion production. This will be in close association with participants #2 and 3.
Participation to workpackage #3. Generation of a complete library of tagged nematode genes. In terms of resources, this workpackage will be the most demanding. Production of the library will be carried out in close participantship with the Ewbank laboratory (participant #3). The first steps of insertion production will be done in the Ewbank laboratory, thus taking advantage of the worm sorter facility, and the remaining steps, as well as the freezing and maintenance of the strains will be carried out in the Segalat laboratory (see section B6 for the detailed description of the operations).
The laboratory will also be in charge of the management and distribution of the strains to other laboratories, and of the public cataloging of the library (project web site and insertions in public databases such as Wormbase).
Expertise/Resources: Our laboratory has been a pioneer in proposing the concept of a public and comprehensive library of transposon insertions in C. elegans (C. elegans International Worm meeting, 1999). Since 1999, we have explored the feasibility of this approach through various pilot experiments, first with endogenous Tc transposons (Martin et al. Genetics, 2002), and more recently with the Mos1 system. During the course of these experiments, we have gained knowledge and know-how on how to handle the strains. Our laboratory has optimized the protocols of inverse PCR and subsequent purification for sequencing now used by several laboratories. A pilot experiment resulting in approximately 1000 Mos1 insertions will be completed shortly and will provide important data about the distribution of the insertions in the genome and how mutagenic the insertions are. The analysis of this data is part of the Workpackage #1. Most of the financial resources allocated to our laboratory will be used to the generation of the library. It must be emphasized that the cost of insertions may vary during the course of the project. Budget projections are based on the best possible estimates at the time of the application (March 2003) and calculated for 30,000insertions. Potential cost optimization will be used to increase the number of insertions produced within the budget allocated.
Complementarity with other participants: The Ewbank and Segalat laboratories already collaborate and have strong interactions prior to the start of the proposed project. The Bessereau and Segalat laboratories have close links on the Mos1 systems and upgrades. The Bessereau laboratory has been instrumental in implementing the Mos1 system in the Segalat lab and, conversely, experience gained from pilot experiments run in the Segalat lab has been a valuable source of data for optimizing the system.
Publications relevant to the project.
1. Jospin M, Jacquemond V, Mariol MC, Segalat L, Allard B. (2002) The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans. J Cell Biol. 159: 337-348.
2. Jospin M, Mariol MC, Segalat L, Allard B. (2002) Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans. J Physiol. 44: 373-384.
3. Martin E, Laloux, H, Couette G, Alvarez T, Bessou C, Sookhareea S, Labouesse M, Ségalat L (2002) Identification of 1088 new transposon insertions of C. elegans, a pilot study towards large-scale screens. Genetics, 162, 521-4.
4. Mariol MC, Ségalat L. (2001) Muscular degeneration in the absence of dystrophin is a calcium-dependent process. Current Biology, 11, 691-4.
5. Gieseler K., Grisoni K. and Ségalat L. (2000) Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans. Current Biology, 10, 1092-1097. (see also Current Biology, 10, R795-797 for comments).