Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange...

I outline a streamlined method to insert large, single-copy transgenes into the Caenorhabditis elegans genome using recombination-mediated cassette exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (>1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, and unc-119) that yield lines expressing different colored FPs (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell-specific promoters tested. However, in certain orientations, promoters exhibit crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone, and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rapid RMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.