Participant 2. Jean-Louis Bessereau (JLB), MD, PhD
INSERM U. 497, Ecole Normale Supérieure, France
General: The focus of our laboratory is the analysis of synapse formation and function using a genetic approach in C. elegans. Specifically, we are conducting several pharmacological screens using Mos1-mediated mutagenesis, a new technology that was initially developed by JLB and that we kept improving in our laboratory. The use of Mos1 considerably speeds up the identification of mutant genes and we already identified new genes involved in nicotinic neurotransmission that are currently being characterized. Our group has now a deep experience of the Mos1 system. JLB was the first to succeed in mobilizing a heterologous transposon in the C. elegans germline and his experience will be valuable to the other members of the consortium during the development of new transposon systems.
Role in the project: Our main contribution will be the development of new tools to engineer C. elegans genes based on controllable remobilization of Mos1 insertions. Among the collection of Mos1 insertions that will be generated by the consortium, some insertions will not be mutagenic. However, they will represent extremely valuable resource to further manipulate the locus where they inserted. First, imprecise excision of Mos1 has been demonstrated to cause various lesions such as gene deletions. We showed that controllable Mos1 re-excision is very efficient. By developing a strategy to recover imprecise excision events at high frequency, it will be possible to efficiently knock-out genes at low cost. Second, genomic knock-in techniques based on homologous recombination are not yet available in C. elegans. Mos1 excision causes a double stranded DNA break that can be repaired using a transgene as template. Since the consortium proposes to generate Mos1 insertions in a very large number loci, this strategy would provide a general tool for C. elegans gene knock-in.
Expertise/Resources: JLB was at the origin of the Mos1 system that he developed while working for several years in the laboratory of Dr Jorgensen (University of Utah). Our group in Paris kept characterizing and improving Mos1-controllable transposition. We are routinely using Mos1-mediated mutagenesis in screens and we cloned several genes with this technique (manuscript in preparation). We have also generated numerous GFP markers and antibodies to analyze synapse formation and receptor dynamics. The laboratory is fully equipped for molecular biology, biochemistry, microscopy, microinjection and for culture and maintenance of nematode strains.
The laboratory is implanted in the Biology Department of the Ecole Normale Superieure in Paris, a prestigious university, which attracts the very best students in France. This department contains outstanding research groups especially in development and neurobiology fields. The department provides access to on-site facilities including an imaging platform and a genomic platform with DNA sequencing service. A bioinformatics group has recently joined the department and has strengthened the local computing infrastructure.
Complementarity with other participants: Since the establishment of a joint research contract funded by INSERM (ITM, 2000-2002), JLB has enjoyed a close collaboration with the Ewbank group (participant #3) that has focused on the development of Mos1-related techniques. In the context of the current proposal. Mos1 insertion generated by the Segalat group will be used as starting material for development of knock-out and knock-in strategies. The specific knowledge of the Mos1 system will be shared with participants 3, 5 and 6 for the generation of large-scale insertional mutagenesis (WP3). Preexisting experience with heterologous transposons will be shared with participants 1 and 3 for the development of new transposon systems (WP2). Pharmacological screens will be used to evaluate the mutagenicity of transposons in the large-scale insert collection generated in WP3. Experience in scoring behavioral phenotypes will be valuable for evaluating the automated platform developed by participant 4 (WP5).
Main publications relevant to the project:
1. Bessereau, J.-L., Wright, A., Williams, D., Schuske, K., Davis, W., & Jorgensen, E. (2001) Mobilization of a drosophila transposon in the C. elegans germline. Nature, 413, 70-74.
2. Gally, C. and Bessereau, J.-L. GABA is dispensable for the formation of junctional GABA receptor clusters in C. elegans(2003) J. Neurosci. 23: 2591-2599.
3. Bosher, J.M., Hahn, B.S., Legouis, R., Sookhareea, S., Weimer, R.M., Gansmuller, A., Chisholm, A.D., Rose, A.M., Bessereau, J.-L., Labouesse, M. (2003) The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces. J Cell Biol. 161: 757-768.