MultiSite Gateway (Invitrogen Life Technologies) is a development of the conventional Gateway recombination system but allowing multiple DNA fragments to be joined together. The applicability of this novel system to the cloning of C. elegans promoter fragments upstream of reporter genes was explored. The MultiSite Gateway system would allow parallel re-cloning both of C. elegans promoter fragments from promoter entry clones into other plasmid vectors, for genomic investigations of promoter activities, and of other protein coding regions from Gateway ORF entry clones downstream of characterized C. elegans promoters. Six promoter fragments, for C. elegans genes B0464.4, F44B9.2, F54D5.1, F56D5.8,
pes-1 and
fkh-2 that we had previously fused to a lacZ reporter, were selected for re-cloning upstream of a gfp reporter in a test of the MultiSite Gateway system. Promoter fragments were amplified by PCR from C. elegans total genomic DNA, using primers containing the appropriate Gateway recombination sites (B4 for the upstream primer and B1 for the downstream primer), and cloned using the Gateway BP reaction. A gfp entry clone was generated with a gfp reporter gene flanked by B1 and B2 recombination sites, as for C. elegans ORF entry clones. Each promoter entry clone was then recombined with the gfp entry clone and a vector plasmid in a three-molecule, Gateway LR reaction. This generated plasmids containing reporter gene fusions with the arrangement B4-promoter-B1-gfp-B2, which were used in transformation of C. elegans for examination of GFP expression in transgenic lines. The 24bp B1 recombination site, between the promoter and reporter, did not interfere with GFP expression in C. elegans. These experiments relied on relatively simple techniques and the efficiencies observed suggest scaling up for genomic analysis of C. elegans promoter activity should be feasible.