Feasibility of Genome-Scale Construction of Promoter::Reporter Gene Fusions for Expression in Caenorhabditis elegans Using a MultiSite Gateway Recombination System

With genomes sequenced, biological investigations are increasingly organized on a genome-wide scale. The strategies followed depend on techniques that can be applied in parallel to a large number of samples, typically one for each gene in a genome. Microarrays are a major example used in the study of gene transcripts, but other aspects of the genome also need to be studied at this scale. The Gateway cloning technology (Hartley et al. 2000) utilizes site-specific recombination to transfer DNA segments between vector backbones. This technology allows genome-wide sets of proteins, the functional products of most genes, as encoded by cloned open reading frames (ORFs), to be studied in multiple ways in a high-throughput mode. Once cloned, the ORFs can be efficiently recloned, in parallel, into numerous alternative plasmid vectors (Destination Vectors), each of which can be designed for expression and study of the protein products with a specific desired technique. The recombination reactions occur in small, λ-derived, recombination sites flanking the DNA segments of interest. As a result, the integrity of the transferred sequence remains intact and resulting fusions can be directly compared. A complete set of Gateway “ORF Entry Clones”, the starting point for recloning into any Gateway destination vector, thereby becomes a highly valuable genomic resource. Such a strategy has been demonstrated for the large scale determination of protein-protein interactions for Caenorhabditis elegans (Walhout et al. 2000). Another major component of the genome that needs to be investigated on a genome-wide scale is the DNA containing the regulatory elements that direct the appropriate temporal and spatial expression of the genes as an organism develops or responds to external stimuli. The genome of C. elegans is relatively densely packed for a metazoan (The C. elegans Sequencing Consortium 1998) and transcriptional regulatory elements, including enhancers, tend to be close upstream of the translational initiation codon. These upstream regions could be the targets of Gateway cloning, in preparation for parallel study through recloning into alternative Destination Vectors designed for studying promoter function through different experimental approaches. Further advancements in the Gateway Cloning technology have provided an expanded collection of recombination sites, each with unique specificities (Cheo et al. 2004). By developing strategies that employ three or more unique recombination site specificities, two or more DNA segments can be linked with high efficiency in an order and orientation-specific way. Using this approach, a collection of Promoter Entry Clones and a collection of ORF Entry Clones could be generated such that they could be efficiently fused in any combination. Although there are other recombination-based cloning technologies, in which the recombination reactions are carried out in vitro (Liu et al. 1998) or in vivo either in Escherichia coli (Court et al. 2002) or in Saccharomyces cerevisiae (Winzeler et al. 1999), compatibility with the established library of Gateway ORF clones for C. elegans (Walhout et al. 2000; Reboul et al. 2001) would be a significant advantage. The feasibility of using MultiSite Gateway technology for cloning C. elegans promoters is explored here.