Screening procedure for the creation of new insertions and identification of lethal and sterile mutations. (A) P1 cross: Mass-crosses were made between the donor (EGFP-marked eyes) and the helper strain (DsRed-marked eyes). The donor strain has an additional insertion site-dependent, muscle-specific enhancer-trap pattern. (B) P2 cross: Single hybrid females carrying both the donor and helper elements (simultaneous expression of EGFP and DsRed) were crossed to three pearl males. (C) F1 cross: A single individual carrying a stable new insertion was selected from the offspring of a P2 cross and crossed to several pearl mates. A remobilization event was evident in beetles that still showed EGFP-marked eyes, but had lost the muscle-specific enhancer-trap pattern. Note the altered enhancer-trap phenotype of the new insertion line in this example (EGFP expression in the wings). (D) F2 cross: All EGFP-marked offspring of the F1 cross were heterozygous for the insertion and were sibling-crossed. (E) F3 cross: Several single-pair matings were set up. (F) Test for lethality and sterility: Marker gene expression of the offspring of each single-pair mating was evaluated to determine whether their parents had been hetero- or homozygous for the piggyBac insertion (see Methods). Each single-pair mating was assigned to one of five classes (small black arrows; Table 4). The combined evaluation of all single-pair matings was used to define the phenotype of the insertion (see Table 5). (G) Summary of all identified insertions. For further details see methods part "Generating new piggyBac insertions", Table 1, and text.