An efficient negative selection marker for Mos1-mediated single-copy integration in Caenorhabditis elegans

In C. elegans, Mos1 transposition has been successfully coopted for the insertion of single copies of transgenes at predetermined chromosomal loci (Frokjaer-Jensen et al. 2008; Frokjaer-Jensen et al. 2012). This method, known as Mos1-mediated single-copy integration (MosSCI), employs C. elegans strains that contain the Mos1 element at selected chromosomal loci (Vallin et al. 2012). The desired transgene is constructed on a plasmid vector such that it is flanked by sequences homologous (homology arms) to either side of the existing Mos1 element on the chromosome. The transposase expressed from another plasmid that is introduced along with the transgene-carrying plasmid swaps the Mos1 element on the chromosome with the transgene, which results in single-copy integration at the specific locus. The C. elegans strains used are homozygous for the ed3 allele of unc-119(ed3) as well, which renders them locomotion-defective. The transgene plasmids contain a wildtype copy of cbr-unc-119 which is cointegrated along with the transgene during the MosSCI event. This restores wildtype locomotion, and thus serves as a positive selection for successful insertion of the transgene. The plasmids are introduced by microinjection into the germ line. Plasmids thus introduced often join in tandem and form of extrachromosomal arrays which are transmitted in a non-Mendelian fashion (Stinchcomb et al. 1985). Due to this, in addition to the integrated copy, transgene expression can result from the extrachromosomal array as well. Consequently, the positive selection based on unc-119(-) rescue is insufficient to identifying animals that carry the integrated transgene but array-negative. To circumvent this, additional plasmids that carry transgene reporters without the homology arms are included for negative selection—only the array-positive animals express these reporters (Frokjaer-Jensen et al. 2008).