A set of ~400 overlapping Pacman BAC clones was used to create small duplications (average length 88 kb) covering most of the 22-Mb sequenced portion of the X chromosome. Using the Pacman system (FBrf0194996), the BAC clones were inserted into an attP docking site on chromosome arm 3L; available as homozygous or balanced heterozygous stocks.
Docking-site insertion on 2L used for one line.
Note concerning progenitor docking site: PBac{y+-attP-3B}VK00037 (on chromosome 2L) was used for one line in this collection.
A tiling path of Pacman BAC clones spanning the sequenced portion of the X chromosome was selected from the CHORI-321 library. Using FlyBase gene annotations, clones were selected to maximize coverage of complete gene annotations and unannotated 5′ control regions. For some regions not covered by the CHORI-321 library, clones from the CHORI-322 library (with smaller insert size) were selected. The final selection of 566 verified clones had an average insert length of 87,700 bp and an average overlap of 47,800 bp. For a small number of genes and regions clones were not recovered; these are unnumerated in Table S1 ofFBrf0212670. Using the Pacman system (FBrf0194996), BAC clones were injected into embryos and recovered as integrants in the attP site PBac{y+-attP-3B}VK00033 on chromosome arm 3L. Multiplex PCR confirmed that for 382 clones (94%) the transgenic BAC had integrated into the proper docking site; of these, 2 independent lines were recovered for 214 (56%). Transgenic lines were tested for homozygous viability and fertility; homozygous lines were established whenever possible.
In terms of clones injected, efficiency of transformation was 66% upon a first injection attempt; 80% after a second injection attempt. In terms of animals injected, average transformation efficiency was one transformant-producing fly for every 54 fertile G0 animals. Of 461 BAC clones injected, transgenics were recovered for 408 of them (88%). Loss of the mini-white marker has not been observed over many generations, indicating that the duplications are stably maintained.
