One of the biggest challenges of the post-genomic era is to describe the gene expression patterns and protein-protein interactions on genome wide scale. Protein tagging has been used extensively for localization and complex purification in yeast, where homologous gene targeting is straightforward. To extend this research to Caenorhabditis elegans we have developed a method for fast and efficient gene tagging using homologous recombination in bacteria.The region of interest is subcloned in a shuttle vector from genomic BAC clone and the tag is inserted precisely where desired. Transgenic worm lines are then generated by ballistic transformation. Large DNA sequences can be modified in this way and the method is applicable even for very long genes. Preserving the endogenous genomic context ensures exact reconstitution of the time, space and level of expression of the wild type gene. We designed and tested series of subcloning vectors containing the
unc-119 gene, a low-copy replication origin and a selectable marker for bacteria. The tag is added as a cassette with selection marker, which is flanked by frt sites and can be removed upon expression of Flp. We have generated targeting cassettes for several GFP variants and double affinity tags.Caenorhabditis briggsae genomic BAC clones are available from the BACPAC Resource Center, Oakland, California. To map the clones we wrote a program that BLASTs the BAC end sequences to the genome, filters the highest scoring matches and exports them in a format that can be visualized with the genome browser of Wormbase. Use of these BACs is not limited to C. briggsae as most proteins are highly conserved between the different Caenorhabditis species. On DNA level however, the genes are divergent enough to allow specific knockdown by RNAi of the endogenous, untagged allele. This opens an interesting opportunity for comparative cross-species studies.To confirm the validity of our approach we have tagged several proteins with known expression patterns and protein-protein interactions. Data will be presented on the GFP localization and protein complex purification of the Trithorax group protein
lin-59.