Progenitor stocks were isogenic FRT-carrying transgenic insertion lines, using insertions of P{RS3} and P{RS5}. Initially, internal recombination mediated by FLP recombinase between the FRT sites in P{RS3} and P{RS5} was induced (flip-out), to produce the remnant form of these elements, P{RS3r} and P{RS5r}, each of which has a nonfunctional white gene and a single FRT site. The flip-out recombination event was extremely efficient, over 98%. Recombination between FRT elements in two insertions in the appropriate relative orientation results in deletion of the genomic region between the insertions and production of a hybrid element that contains one FRT element at the recombination point (FBrf0091061). FLP-mediated recombination between P{RS3r} and P{RS5r} elements (flip-in) in trans and in the appropriate orientation produced a hybrid P-element with two 3' ends, a reconstituted functional miniwhite gene, and a deletion of the intervening genomic region. The efficiency of the flip-in event varied with the size of the deletion produced.

Deletion-generation strategy made use of extensive collection of ~3300 isogenic FRT-carrying transgenic insertion lines (FBrf0179424); all lines were mapped by inverse PCR. The pilot experiment described in FBrf0179424 was scaled up. Deletion endpoints were confirmed by PCR (with primers within the predicted flanking genomic DNA and/or within the residual P-element at the recombination point), by genetic complementation, or by both.

To confirm the stability of the double 3′ P-elements, the loss of the miniwhite marker in the presence of P-transposase was assayed for 3 deletion lines; no miniwhite[-] progeny were observed.

Of 870 different deletions that were attempted, 665 (76%) were recovered. This set covers ~77% of the euchromatic genome; average size of 368kb; average of 44 genes uncovered. Of the 209 deletions in the core collection, 89% have been confirmed by PCR, 64% confirmed by genetic complementation, and 54% by both methods.