Figure 1 Overview of new RMCE landing sites:A) Structure of the miniMos and CRISPR/cas9 mediated single and two-component landing sites. Coding regions and Mos1 transposase arms (thick rectangles), promoters (thin rectangles), and recombinase sites (triangles) are labelled. Unlabeled thick arrows represent 3' UTRs:
unc-54 (yellow),
gpd-2/3 (grey),
his-58 (blue),
glh-2 (pink). The unlabeled grey region between GFP-C1 and
his-58 is a flexible linker. loxP, FRT, and FRT3 sites (triangles) not drawn to scale for clarity.B) Position and structure of the genomic interval of landing sites. jsSi1579 is a two-component landing site integrated at a
cas9 sgRNA site just adjacent to the position of the widely used
ttTi5605 Mos1 insertion on Chr II. jsSi1691 is a single-component landing site integrated at the same position as jsSi1579. jsSi1669 is a single-component landing site integrated at a
cas9 sgRNA site just adjacent to the position of the cxTi10882 Mos1 insertion on Chr IV.
js1570 is a derivative of jsTi1453 on Chr I in which the miniMos left arm was deleted using CRISPR/cas9. Mos1 insertions are represented by solid blue triangles. The landing site insertions are represented as triangles colored with a purple gradient oriented such that the dark side of the gradient represents the 5' end of GFP-
his-58 coding sequences within the insertion and the light side represents the 3' end. The position of the region on the chromosome (bp) is listed just below the line representing the chromosome. Coding genes are represented in pink and teal and non-coding genes in grey. Analogous schematics for the previously characterized landing sites are also available [See figure S2 of Nonet, (2020)].C) Comparison of the expression levels of identical insertions at various RMCE landing sites. Expression level of a
mec-4p GFP-C1
tbb-2 3' construct integrated at the previously described landing sites (Nonet, 2020) and the new Chr I, Chr II and Chr IV landing sites. I assumed that integration into jsSi1579 and jsSi1691 would yield the same expression level as they yield molecularly virtually identical insertions. The Chr II integration was made using jsSi1579. Removing the mosL arm from jsTi1453 has only a minor effect on expression of an integrated tetO 7X GFP reporter (Rp) when driven by a
mec-4p Tet OFF driver (Dr). Direct GFP fusion and bipartite reporter strains were imaged and quantified under the same conditions and thus can be directly compared. Strains imaged: NM5196, NM5209, NM5228, NM5236, NM5337, NM5467, NM5580, NM5582 and NM5633. n=14-21 for ALM and n=28-42 for PLM.D) Images of the gland cell background GFP expression of transgenic animals carrying tetO 7X GFP-C1 integrated into jsTi1453 and the &#
x2206;mosL
js1570 derivative. Strains imaged: NM5264 and NM5327. Scale bar: 20 µm.E) Frequency of insertions obtained at distinct landing sites. All injected animals were counted regardless of perceived quality of injection or survival. In most cases only a single gonad was injected. F1 Rol progeny were typically grouped 6 per plate for identification of integrated lines. Lines represents the number of independent F1 progeny plates that segregated an integrated line. Success is defined as obtaining an integration event at the expected genomic position. The insert size does not include the 7.85 kb vector and SEC sequences excised during heat shock. § Injections performed using PB washed DNA.