1. Cell Biology
  2. Neuroscience
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A genetic toolkit for tagging intronic MiMIC containing genes

  1. Sonal Nagarkar-Jaiswal
  2. Steven Z DeLuca
  3. Pei-Tseng Lee
  4. Wen-Wen Lin
  5. Hongling Pan
  6. Zhongyuan Zuo
  7. Jiangxing Lv
  8. Allan C Spradling
  9. Hugo J Bellen  Is a corresponding author
  1. Howard Hughes Medical Institute, Baylor College of Medicine, United States
  2. Howard Hughes Medical Institute, Carnegie Institution for Science, United States
  3. Baylor College of Medicine, United States
  4. Texas Children's Hospital, United States
Research Advance
Cite this article as: eLife 2015;4:e08469 doi: 10.7554/eLife.08469
3 figures and 1 additional file

Figures

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.

https://doi.org/10.7554/eLife.08469.002
Figure 1—source data 1

List of constructs.

https://doi.org/10.7554/eLife.08469.003
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
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.

https://doi.org/10.7554/eLife.08469.005
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. (AI, except G), 25 μm (G). Eys, eyes shut.

https://doi.org/10.7554/eLife.08469.006

Additional files

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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