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.
Using full-length coding sequences from various sources, ORFs were cloned into a Gateway vector by a two-step PCR strategy: a first round with gene-specific primers which incorporate a Kozak sequence, then a second round using attB primers. After verification of an intact sequence, ORF cassettes were introduced into pGW-HA.attB plasmids which contained randomized barcode sequences. The resulting vectors carry the ORF of interest preceded by Scer\UAS sequences, flanked by mutated Scer\FRT sites, and followed by a C-terminal 3xHA tag; a Sliv\attB recombination site is present downstream of the HA tag. 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 on the X-chromosome. Surviving males were mated in pairs; stocks were established using single F1 males, which were subsequently used for single-fly PCR and sequencing to identify the barcode. Lines representing recurring barcodes were discarded.
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.
Epitope-tagging may interfere with protein function; the flexible linker included in these constructs is thought to minimize steric interference. Addition of an Scer\FRT site between the ORF and the 3xHA epitope tag allows exchange of the C-terminal sequences, including the epitope tag.
