The rapid RMCE landing sites were created in two steps using RMCE instead of using either miniMos or CRISPR as with other RMCE landings (Figure 1). One can also create novel rapid RMCE sites using CRISPR.
The advantage of this modular approach is that it enables one to create a customized landing site that expresses any fluorescent marker under the control of any promoter as the basis for visualizing the landing site and subsequently tracking the insertion. One starts with an intermediate landing site (available presently for Chr I at jsTi1453, Chr II adjacent to ttTi5605, Chr IV adjacent to cxTi10882 and Chr V adjacent to oxTi365). The promoter of choice, an FRT site, the fluorescent marker of choice and a 3′ UTR are then co-inserted into the landing site vector pLF10F3ShCmx5F ([map], [sequence]) using a SapI GG assembly reaction to create the RMCE integration vector. One then uses the new vector to create the novel landing site (Figure 2).
Last edited 5-9-2022