It has been argued that, to globally understand development, it will be crucial to generate databases describing where (in what cell) and when (at what stage of development) each protein is expressed. One possible approach is to construct transgenic C. elegans strains carrying reporter genes, such as that encoding the Green Fluorescent Protein (GFP), driven by particular promoters and examine expression patterns throughout development. Such studies have already been successfully carried out with relatively small subsets of genes, this project aims to address conceptual and technical issues that will allow us to apply this approach to a genome wide scale. We propose to clone the majority of worm promoters into a novel Gateway compatible vector. The availability of a comprehensive collection of cloned worm promoters (promoterome) will be very useful to the scientific community. We use an innovative Gateway technology with which we will transfer cloned promoters into various destination vectors. Each promoter can then be used to detect expression of reporter genes, or to drive expression of their cognate ORFs, fused to either GFP, or any other Tag-encoding sequence. These can be used to examine sub-cellular protein localization, for complex purifications or in genetic rescue experiments. To increase the throughput of generating transgenic worms to a genome scale, we will adapt the recently developed ballistic transformation protocol (Praitis, 2001) This method allows genomic integration of low copy plasmids which may allow more physiological regulation of the transgenes than extrachromosomal arrays. We will then use a semi-automated 96 well confocal microscopic platform (Atto Bioscience) for rapid imaging and analysis of multiple independent transgenic strains. All the data generated will be made available to the community on a freely accessible database. To illustrate usage of Multisite Gateway to generate promoter::ORF::GFP fusion proteins. We created a construct in which a Histone2B (B0035.8) is expressed from the
myo-2 promoter, which drives expression in the muscle cells of the pharynx. We then used the microparticle bombardment technique to integrate this construct into the C. elegans genome. As expected, the
pmyo-2::H2B::GFP construct expressed in the muscle cells of the pharynx, and the H2B::GFP fusion localized to the nucleus.