See additional related dataset FlyORF-3, an epitope-tagged UAS-ORF collection.
A collection of stocks of UAS-ORF transgenic recombinants that carry the ORF of interest marked by a C-terminal HA epitope tag and preceded by Scer\UAS sequences, Hsp70 promoter sequences and a Kozak consensus sequence for efficient translation. All constructs also carry the w+mC mini-white marker, and all have been introduced by recombination into a site at 86F on the third chromosome. Controlled expression of the ORFs can be effected by introduction of Scer\GAL4 drivers. See also the related dataset, an untagged UAS-ORF collection, FlyORF-1.
Full-length ORFs were cloned from cDNAs from the BDGP Drosophila Gene Collections (releases 1-3) or from the SD_cDNA library (Berkeley Drosophila Genome Project). ORFs were cloned by PCR with gene-specific primers then introduced into Gateway-based vectors that contain randomized ‘barcode’ sequences. The barcoded ORF cassettes were transferred to pUASg-HA.attB by a Gateway recombination reaction; the resulting vectors carry the ORF of interest with a C-terminal 3xHA tag preceded by Scer\UAS sequences, plus a Sliv\attB recombination site. Pools of ORF clones were injected into embryos of a strain carrying M{3xP3-RFP.attP}ZH-86Fb (a phiC31\attP recombination target site on the third chromosome), and a source of integrase. Surviving males were mated, then subjected to single-fly PCR amplifying the plasmid region containing the barcode, followed by sequencing. Each new line determined to carry a transgenic recombinant was balanced: repeatedly occurring lines were discarded.
Although epitope-tagging may interfere with protein function, a tagged library holds major advantages over an untagged library: (1) a single antibody can be used to detect any ORF; (2) cross-reaction with related proteins can be avoided, as an antibody specific to the tag can be used; (3) the tagged protein can be distinguished from the endogenous, untagged protein; and (4) immunochemistry becomes possible for even poorly immunogenic proteins or proteins that lack a specific antibody.
