A transposon insertion mutant bank of C. elegans The nematode Caenorhabditis elegans is a model system for the genetic analysis of development and neurobiology. The sequencing of the C. elegans genome is proceeding so rapidly that an (almost) complete genome sequence can be expected in 4-5 years. The challenge for the research community will be to connect biology to the mass of data generated by genome sequencing. The ultimate goal is to understand phenotype in terms of genotype, and the approach will be to establish as many phenotype-genotype relations as possible. This requires an efficient method for reverse genetics of the organism, which is the goal of this project. A frozen library has been established consisting of 8,000 C. elegans lines mutagenized by Tc1 transposon insertion, and a corresponding set of genomic DNA preparations available for inspection by PCR (using primers specific for the transposon and a gene of interest). The library serves as a resource for the research community, and provides insertion alleles to laboratories all over the world.
The aims of this application are the following: 1. Screening of the library will be accelerated, to keep up with the demand for insertion alleles. 2. We will then implement a new screening strategy: flanks of Tc1 elements in each culture will be PCR amplified and gridded on filters. These can be hybridized with a gene specific probe. If a given culture contains an insertion in a gene of interest, this will result in a hybridization signal. This new method has the advantage that filters can be distributed to other laboratories who can do their own search of the library and then request the line of interest. 3. Many Tc1 insertion alleles do not inactivate a gene, but we found that they can be used to isolate derivative progeny in which the transposon plus much of the gene has been deleted. The process of Tc1-induced deletions will be better defined: frequency of deletions, size distribution of the deleted regions, endpoints of the deletions, etc. A mutant library will allow the C. elegans field to make optimal use of the available genome sequences. Workers in other fields may decide to study their gene of interest in this model organism if they find out that gene sequences plus mutant alleles are readily available. Thus this research will be of general importance for developmental biology, neurobiology, and disease oriented research.
Showing the most recent 10 out of 17 publications
