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. 2003 Jun 24;100(13):7720-5.
doi: 10.1073/pnas.1230526100. Epub 2003 Jun 11.

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

Udo Hacker  1 , Sverker Nystedt, Mojgan Padash Barmchi, Carsten Horn, Ernst A Wimmer
Affiliations

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

Udo Hacker et al. Proc Natl Acad Sci U S A. .

Abstract

P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.

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Figures

Fig. 1.
Fig. 1.
(a) piggyBac-based mutator transposon marked with 3xP3-EYFP. (b) Hermes-based jump-starter transposon marked with 3xP3-ECFP.(c) Expression of 3xP3-EYFP and 3xP3-ECFP in unpigmented and pigmented eyes. (Left) Bright-field view of eyes expressing 3xP3-EYFP (upper left) or 3xP3-ECFP (upper right) in a white- background, and eyes expressing 3xP3-EYFP in the presence of one (lower left) or two copies of a white transgene (lower right). (Center) Same as Left viewed through the EYFP filter set; 3xP3-EYFP is detectable in white- eyes (upper left) or in eyes expressing one copy of a white transgene (lower left); in eyes expressing two copies of a white transgene (lower right), 3xP3-EYFP is detectable in the ocelli (arrow) and the pseudopupil of the compound eye (not visible in frontal view); 3xP3-ECFP is not detectable (upper right). (Right) Same as Left viewed through the ECFP filter set. 3xP3-EYFP is not detected (left and lower right), but 3xP3-ECFP is detected (upper right).
Fig. 2.
Fig. 2.
(a) Distribution of lethal piggyBac insertions on chromosome 3. Color code represents number of piggyBac insertions isolated for each letter division of the third chromosome. Transposon insertions in red show the 10 genes most frequently hit by P elements (1). Transposon insertions in green show the genes most frequently hit by piggyBac in this screen. dlp, ftz-f1, and CG17370 were hit three times each, and pyd and the intergenic region between CG2022 and corto were hit four times. (b) Insertion preference of piggyBac within a schematic transcription unit.
Fig. 3.
Fig. 3.
Enhancer trap expression patterns associated with piggyBac insertions. Shown are 5-bromo-4-chloro-3-indolylβ-d-galactoside stainings of embryos from piggyBac insertion lines crossed to P{UAS-LacZ}. (a) Line l(3)PL00759 inserted in hb.(b and c) Line l(3)PL00790 inserted in bnl.(d) Line l(3)PL00757 inserted in lab. (e) Line l(3)PL00779 inserted in srp. (f and g) Line l(3)PL00799 inserted in Mpk2. (h) Line l(3)PL00795 inserted in CG10823.(i) Line l(3)PL00724 inserted in CG10522. (k) Line l(3)PL00749 inserted in CG12546. Anterior is to the left, dorsal side up.
Fig. 4.
Fig. 4.
Embryonic phenotypes associated with piggyBac insertions. (a) Cuticle phenotype of a homozygote of line l(3)PL00779 inserted in srp. (b) Embryo homozygous for line l(3)PL00790 inserted in bnl stained with 2A12 antibodies. (c) Cuticle phenotype of an embryo derived from a female carrying germ-line clones of line l(3)PL00784 inserted in 14-3-3 crossed to a wild-type male. (d) Cuticle phenotype of embryo maternally and zygotically mutant for line l(3)PL00739 inserted in ftz-f1.
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