A genetic toolkit for tagging intronic MiMIC containing genes
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
- Howard Hughes Medical Institute, Carnegie Institution for Science, United States
- Baylor College of Medicine, United States
- Texas Children's Hospital, United States
Figures
Figure 1
Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.
(A) Flp mediated generation of circular DNA with tag sequences. Donor line carries a source of Flp recombinase and phiC31 integrase, and the core cassette for a specific phase (0, 1, or 2) encoding a (GGS)4 flexible linker (L), multiple tags EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF), another (GGS)4 flexible linker, and a splice donor (SD), which is flanked by two inverted attB and two FRT sites oriented in that same direction. Upon Flp expression, the core is flipped out as a circular DNA containing one FRT site. (B) In vivo integration of tag sequence into Minos-Mediated Integration Cassette (MiMIC) locus. At a MiMIC locus in a coding intron, the MiMIC gene trap cassette is replaced by L-EGFP-FlAsH-StrepII-TEVcs-3xFlag-L sequence by phiC31 integrase resulting in loss of yellow+ marker.
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Figure 1—source data 1
List of constructs.
- https://doi.org/10.7554/eLife.08469.003
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Figure 1—source data 2
List of fly strains generated.
Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.
- https://doi.org/10.7554/eLife.08469.004
Figure 2
Crossing scheme for generating EGFP tagged MiMIC lines.
Females carrying the hs-FLP and vasa-phiC31 integrase on the X-chromosome, and the FRT flanked multiple tag (GFSTF) cassette on the II-chromosome are crossed to males carrying a MiMIC insertion in a coding intron of a gene on the III-chromosome. The resulting embryos are heat shocked, and adult progeny with the desired genotype are crossed with flies carrying appropriate balancers. The resulting progeny is screened for the loss of white+ and yellow+ and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.
Figure 3
Expression of EGFP tagged protein in various tissues.
(A) Colocalization of GFP tagged proteins with gene specific antibodies: anti-GFP signals (green) show colocalization with anti-Eys (red) in inter-rhabdomere space in adult eye (top panel) and anti-Delta (blue) in L3 eye imaginal disc in bottom panel. Scale bars, 2 μm and 5 μm. (B) Examples of EGFP expression pattern in different tissues from third instar larvae: brain; l(2)gl (A), Dl (B), twins (tws) (C), Sap-r (E), Rgk3 (F), and Hrb98DE (G), wing imaginal disc: kay (D), hindgut: CG10086 (H), and cells of the cuticle: CG5656 (I). Scale bars 100 μm. (A–I, except G), 25 μm (G). Eys, eyes shut.
Additional files
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Supplementary file 1
Total EGFP tagged genes created: MI: MiMIC insertion, GT: Gene Trap, PT: Protein Trap, Y/N: Yes/No, and L/V: Lethal/Viable
- https://doi.org/10.7554/eLife.08469.007
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A genetic toolkit for tagging intronic MiMIC containing genes
eLife 4:e08469.
https://doi.org/10.7554/eLife.08469
