To facilitate a systematic investigation of C. elegans expression patterns and interactions, we have developed Gene CATCHR (Gene Cloning and Tagging for C. elegans using yeast Homologous Recombination). Gene CATCHR is a novel method of generating reporter constructs that exploits yeast homologous recombination to subclone and tag worm genes. The cloning and tagging steps require PCR, two yeast transformations, and several platings on selective media. Each step is relatively straightforward, and amenable to high throughput techniques. Using Gene CATCHR we can produce functional tagged transgenes that provide accurate information about the spatiotemporal pattern of gene expression and subcellular localization of gene products. Major advantages of Gene CATCHR include:1) Clone size potential: we can capture large genomic regions facilitating the isolation of regulatory sequences that may be important for gene expression. We have used Gene CATCHR to clone 5 operons, up to 20 kb in size. Notably, we have successfully cloned and tagged a 36 kb genomic fragment containing the tra-1 gene.2) Flexibility: any tag can be inserted at any position in a gene without introducing extraneous sequence. Our system can also be exploited to create targeted mutations.3) Scalability: we can clone and tag many genes at once in 96 well format. To date, we have cloned 68 genes, 22 of which have been tagged with GFP. In order to generate a high throughput system, we are currently developing a bioinformatics tool.4) Expression and Function: The expression patterns generated from Gene CATCHR derived transgenes are consistent with those previously reported, and we have documented additional uncharacterized expression patterns. Our transgenes are functional in rescue assays; as proof of principle, we have rescued 5 mutants.All Gene CATCHR related protocols and reagents will be made available to the worm community through our website, www.genecatchr.org (stay tuned!). The expression data generated will also be submitted to WormBase. Gene CATCHR represents an efficient, highly versatile, and powerful system for functional genomic applications.

Affiliation:
- Department of Molecular & Medical Genetics, University of Toronto, Toronto, ON, Canada