Efficient Transgenesis in Caenorhabditis elegans Using Flp Recombinase-Mediated Cassette Exchange

The application of CRISPR technology has greatly facilitated the creation of transgenic Caenorhabditis elegans lines. However, methods to insert multi-kilobase DNA constructs remain laborious even with these advances. Here, I describe a new approach for introducing large DNA constructs into the C. elegans genome at specific sites using a combination of Flp and Cre recombinases. The system utilizes specialized integrated landing sites that express GFP ubiquitously flanked by single loxP, FRT, and FRT3 sites. DNA sequences of interest are inserted into an integration vector that contains a sqt-1 self-excising cassette and FRT and FRT3 sites. Plasmid DNA is injected into the germline of landing site animals. Transgenic animals are identified as Rol progeny, and the sqt-1 marker is subsequently excised with heat shock Cre expression. Integration events were obtained at a rate of approximately one integration per three injected F0 animals-a rate substantially higher than any current approach. To demonstrate the robustness of the approach, I compared the efficiency of the Gal4/UAS, QF (and QF2)/QUAS, tetR(and rtetR)/tetO, and LexA/lexO bipartite expression systems by assessing expression levels in combinations of driver and reporter GFP constructs and a direct promoter GFP fusion each integrated at multiple sites in the genome. My data demonstrate that all four bipartite systems are functional in C. elegans Although the new integration system has several limitations, it greatly reduces the effort required to create single-copy insertions at defined sites in the C. elegans genome.