MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

Abstract

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

Figure 1. The MiMIC transposon system

(a) MiMIC consists of two Minos inverted repeats (L and R), two inverted ΦC31 attP sites (P), a gene trap cassette consisting of a splice acceptor site (SA) followed by stop codons in all three reading frames, and the EGFP coding sequence with a polyadenylation signal (pA), and the yellow⁺ marker. The sequence between the attP sites can be replaced via RMCE, resulting in two attR sites (R). (b) Three attB plasmids for RMCE: a correction plasmid consisting of a multiple cloning site, a gene-trap plasmid consisting of a SA fused to a downstream effector, and a protein-trap plasmid consisting of a reporter flanked by SA and SD sites. (c) Various MiMIC insertions in a hypothetical gene with regulatory element (white), 5' and 3' untranslated regions (grey), and coding regions (black), that can be used for several applications as indicated.

Figure 2. Binary expression and lineage analysis with MiMIC insertions

(a) Gene-trap cassettes that incorporate the GAL4 or QF transactivators for binary activation, and the Flp recombinase for fate mapping. Live imaging (b, d) and confocal microscopy analysis using an anti-Cherry antibody (c, e) of the expression domain revealed by GAL 4 inserted in gogo (b,c ) or the caps locus (d,e). The expression domain of MYPT-75D revealed by GAL4 (f) or QF (g) integrated in MYPT-75D. Live imaging of the GAL4 expression pattern revealed by GAL4 inserted in BM-40-SPARC (h). Scale bars are 50 μm.

Figure 3. Protein trapping with MiMIC insertions

(a) For each protein-trap cassette, three versions were constructed corresponding to the three intron phases (0, 1 and 2). (GGS)₄, flexible peptide linker sequence encoding a GlyGlySer quadruplet tandem repeat. (b) Tag and multi-tag cassettes expressing the indicated reporters are shown. (c) A 100 kb genomic region containing CadN is shown. The location of the Mi{MIC}CadN^MI00393 insertion in a phase 0 coding intron is indicated. (d) Integration of a phase 0 EGFP-FlAsH-StrepII-3×Flag cassette (EGFP) in the indicated orientation and intron phase. L, (GGS)4 linker; R, (attR); P0, splice phase 0; P1, splice phase 1; P2, splice phase 2. Scale bars are 50 μm.

Figure 4. Expression analyses of tagged proteins

(a–i) Detection of different protein-trap alleles of three genes using antibodies against several epitopes and diaminobenzidine/peroxidase (DAB) staining. In each panel, the tag or multi-tag is indicated in the lower right; the portion of the tag that was used as the antigen is italicized. (a–c) Expression of tagged CadN during embryonic stage 15: staining with (a) anti-EGFP, (b) anti-EBFP (c) anti-Dendra. (d-f) Expression of tagged Rfx in stage 15 embryos: staining with (d) anti-V5, (e) anti-EBFP (f) anti-Dendra. (g–i) Expression of tagged Tutl in the ventral nerve cord of stage 15 embryos: staining with (g) anti-EGFP, (h) anti-mCherry, (i) anti-Dendra. (j–l) Fluorescent detection of different protein-trap alleles in live stage 17 embryos expressing tagged Rhea: (j) EGFP (k) mCherry (l) TagRFP. (m–r) Co-localization of protein traps and endogenous proteins in stage 15 embyos for Rfx (m–o) and CadN (p–r). Anti-Rfx staining (m), anti-V5 staining (n), and co-staining (o) in an Rfx∷Dendra-V5 trap. Anti-CadN staining (p), anti-mCherry staining (q), and co-staining (r) in a CadN∷mCherry trap. (s–x) Novel expression detected using protein traps. (s,v) Expression of wnd detected using mRNA in situ hybridization. (t,w) Expression of Wnd detected by anti-EGFP staining of an EGFP-FlAsH-StrepII-3×Flag trap and (u,x) anti-Wnd staining during embryonic stages 11 (s–u) and 16 (v–x). Scale bars are 50 μm (a–l, s–x) and 20 μm (m–r).