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Conditional Mutagenesis in Drosophila

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Science  03 Apr 2009:
Vol. 324, Issue 5923, pp. 54
DOI: 10.1126/science.1168275

Abstract

Most genes function at multiple stages of metazoan development, in dividing and nondividing cells. Generating mouse conditional knock-outs (cKO), where a gene can be eliminated in a temporally and spatially controlled manner, is a valuable technique because it allows study of gene function at any stage of life. In contrast and despite the development of many other powerful genetic tools, cKO has thus far been lacking in Drosophila. We combined several recent molecular and genetic technical advances in an approach termed integrase-mediated approach for gene knock-out (IMAGO). IMAGO allows the replacement of any genomic sequence, such as a gene, with another desired sequence, including cKO alleles that can be used to create positively marked mutant cells. IMAGO should also be applicable to other genetic model organisms.

Most genes have diverse functions in development. Therefore, mutating genes in a spatially and temporally controlled fashion, by generating groups of mutant cells or clones in an otherwise normal animal, is important for analysis of gene function at different life stages. Current clonal methods in the fruit fly Drosophila require mitotic recombination and thus cell division (1), thereby precluding the study of gene function during late stages of differentiation or in adult animals, except if using rare temperature-sensitive alleles. In contrast, the conditional knock-out (cKO) method in the mouse allows tissue-specific gene deletion without the use of cell division and permits the assessment of agene's function in diploid but not polyploid cells at any time (2). Furthermore, because cell division is not required, mutant cells do not need to be siblings. Lastly, cKO permits the combination of different mutant alleles for the same gene.

To develop cKO in Drosophila, we used the atonal (ato) gene, which is expressed in both mitotic and postmitotic cells. ato mutants display measurable phenotypes, including failure of neural cells to differentiate and express characteristic marker genes (3). First, we used ends-out homologous recombination, which uses sequence homology to replace genomic DNA (4), to exchange ato with the white eye color marker (atow). atow is flanked by attP integrase sites, creating a ΦC31 integrase-exchangeable cassette (fig. S1, C to F). This allows the replacement of the marker with sequences flanked by complementary attB sites. Next, ΦC31 (5) is used to replace atow with full-length wild-type ato (atoato), rescuing the mutant phenotype (fig. S1, H and I), or with the transcriptional activator Gal4 (atoGal4), creating a reporter of ato expression (fig. S1J). We name this approach integrase-mediated approach for gene knock-out (IMAGO).

To create ato cKO constructs, we replaced the atow with wild-type ato, flanked by type-3 flippase recognition targets (FRTs, F3) and followed by the Gal4 coding sequence (atoFAFG4; Fig. 1A). FRTs allow flippase (FLP)-mediated deletion, and Gal4 enables marking clones with the green fluorescent protein controlled by upstream activating sequences (UAS::GFP). Thus, in cells in which ato is deleted, GFP is expressed. atoFAFG4 rescued ato mutants (Fig. 1B) and, when deleted via an eye-specific FLP, produced ato mutant phenotype (Fig. 1B').

Fig. 1.

(A) Schematic of ato cKO using IMAGO. Genomic (B) rescue and (B′) FLP-mediated induction of ato mutant eyes. (C to E) GFP-marked ato mutant clones in SOP and PRs. Ato mutant SOP fail to differentiate (C), as do large ato mutant clones in the eye (D and D′). Single ato mutant PR differentiate as non-R8 (E to E″). (E′) A basal section through the disc in (E) and (E″) enlargement of (E′), indicating non-R8 axons (arrows).

We tested atoFAFG4 in dividing sensory organ precursors (SOP) of the leg (Fig. 1C) and nondividing photoreceptor neurons (PRs) in the retina (Fig. 1, D and E). None of the emerging SOP (blue) expresses GFP. Therefore, SOP do not develop from ato mutant cells (green). In addition, as previously shown, large ato mutant eye clones fail to express a differentiation marker (red; Fig. 1, D and D′'). The fly retina is a tissue where nonsibling PRs interact. All retinal cells express Ato before differentiation, but the requirement for ato in single postmitotic PRs is unknown. In single-cell IMAGO clones (Fig. 1E), mutant cells (green) never become R8 (blue) as expected but differentiate as non-R8 PRs (red) if neighbored by a wild-type R8 (blue) and send axons toward the brain (Fig. 1, E' and E”).

Generation of IMAGO alleles required 6 to 8 months, in line with the time required for other types of mutations. Although it may prove more challenging for complex loci, the problem should be surmountable by using recombination-mediated genetic engineering (6). Like other clonal techniques, IMAGO relies on existing FLP tools, making its efficiency limited by that of these tools. IMAGO offers two advantages. First, mutant cells are induced in a diploid tissue at any time. For example, specific neuronal circuits can be mutated to study animal behavior or cellular degeneration without developmental effects. Second, the mutant gene can be replaced by any sequence.

Supporting Online Material

www.sciencemag.org/cgi/content/full/324/5923/54/DC1

Materials and Methods

Fig. S1

References and Notes

References

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