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First published online September 15, 2003
doi: 10.1242/10.1242/dev.00774

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Genetic mosaic techniques for studying Drosophila development

Department of Zoology, University of Wisconsin, 250 North Mills Street, Madison, WI 53706, USA e-mail: ssblair{at}wisc.edu

View larger version (26K): [in a new window]   Fig. 1. Chromosome behavior during and after irradiation-induced mitotic recombination. The clones produced by the two homozygotic daughter cells of a mitotic recombination event are shown below. The photograph shows a shaggy mutant clone, which lacks anti-Myc staining (green), and its sister `twin spot' (+/+), which has a double dose of the Myc epitope, in a pupal wing blade.

View larger version (46K): [in a new window]   Fig. 2. The Minute technique. The homozygous mutant cells produced by mitotic recombination lack the Minute mutation, and thus divide more quickly than the surrounding heterozygotic cells. The homozygous Minute clone usually dies. The photograph shows a wild-type clone, lacking GFP (green), in a +/RpS52 [also known as Minute(1)osp] late third instar wing imaginal disc.

View larger version (27K): [in a new window]   Fig. 3. FRT-induced mitotic recombination, catalyzed at FRTs by hs-FLPase. The photograph shows several engrailed clones, lacking anti-Myc staining (green), in a pupal wing blade. FRTs, FLPase recombination targets; hs-FLPase, heat-shock-induced FLP recombinase.

View larger version (23K): [in a new window]   Fig. 4. The FLPout technique. (A) hs-FLPase catalyzes the removal of an FRT, a marker gene and a transcriptional termination signal (Term.) from the FLPout construct, allowing the constitutive promoter to drive the expression of the downstream gene sequence. (B) The photograph shows several clones generated using a FLPout-cubitus interruptus (ci) construct (Hepker et al., 1997), which can identified by the high levels of anti-Ci staining (red), in a late third instar wing imaginal disc. FRT, FLPase recombination target; hs-FLPase, heat-shock-induced FLP recombinase.

View larger version (32K): [in a new window]   Fig. 5. (A) The Gal4 UAS system. A construct that contains a known promoter or enhancer coupled to the Gal4 gene drives region-specific expression of Gal4. Gal4, in turn, stimulates the transcription from a construct that links the UAS sequence to a chosen coding sequence. (B) The Gal4 enhancer trap. When the enhancer trap construct falls near an active endogenous enhancer, that enhancer drives Gal4 expression. (C) The EP construct. In the presence of Gal4, the UAS and promoter in the inserted UAS-EP construct drive the expression of neighboring genes. (D) A late third instar wing disc showing the region-specific expression of UAS-GFP (green) driven by a ptcGal4 enhancer trap. Strong anti-Ci (Cubitus interruptus) staining (red) shows the anterior compartment. (E) In situ hybridization of a late third instar wing disc showing the ectopic expression of an endogenous gene driven using an EP insertion. ptcGal4 drives the expression of crossveinless 2 (cv-2), which is located near the EP(2)1103 insertion. Reproduced, with permission, from Conley et al. (Conley et al., 2000). (F) Generating dsRNA using a UAS-hairpin construct. Gal4 drives the expression of a UAS construct containing two inverted coding regions placed head to head (red, arrows), separated by a short spacer sequence. The mRNA produced by the construct folds back upon itself to form dsRNA, which will interfere with expression of the corresponding endogenous gene.

View larger version (15K): [in a new window]   Fig. 6. Using UAS-FLPase and FLPout constructs to mark the descendents of Gal4-expressing cells. (A) Gal4 drives expression from the UASFLPase construct. The FLPase in turn activates the FLPout construct by deleting the sequences between the FRTs, leading to the irreversible expression of ß-gal in the descendents of cells that expressed Gal4. (B) Anti-ß-gal staining (green), showing the FLPout clones in a late third instar wing disc containing ptcGal4, UAS-FLPase and FLPout-lacZ. Anti-Ci staining (red) labels the anterior compartment. Note the presence of clones in the posterior compartment, which were not present in the ptcGal4 UAS-GFP disc (Fig. 5E). ß-gal, ß-galactosidase; Ci, Cubitus interruptus; FLPase, FLP recombinase; ptc, patched.

View larger version (30K): [in a new window]   Fig. 7. The mosaic analysis with a repressible cell marker (MARCM) technique. Both Gal80 and Gal4 expression is driven using tubulin promoters (tub). In the parent cells, the Gal80 inhibits the activity of Gal4. Mitotic recombination creates a clone of homozygous mutant cells that lack the tub-Gal80 construct. The Gal4 generated by the tub-Gal4 construct (purple) is now free to drive expression from the UAS-gene construct (blue).

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