Transgenic animals were designed to carry a short hairpin RNA (shRNA) under UAS-GAL4 control. To design the shRNA for any given gene, sequence for all exons, or all exon portions common to all transcripts, were reverse complemented and all possible 21-bp subsequences were determined. Sequences with off-target matches of 16-bp or more were discarded. The remaining sequences were scored as per Vert et al., 2006 (PMID: 17137497). Top and bottom strand oligonucleotides for the top scoring sequence were cloned into pVALIUM20, which by virture of it's hsp70 basal promoter, drives excellent knockdown in the soma and works well in the female germline. The hairpin-containing transgenes were inserted via site-specific recombination into genomic loci known to be optimal for expression, either the P{CaryP}attP2 or P{CaryP}attP40 target element.

shRNA sequences were predicted for all annotated genes, the vast majority using 'designer of small interfering RNA' (DSIR, Vert et al., 2006, PMID: 17137497). To minimize off­target effects, shRNAs whose guide or passenger strands had complementary matches of 16 nucleotides or more to the fly transcriptome. Only shRNAs that lacked sequence complementarity to annotated microRNA ‘seed’ sequences were considered. Hairpin constructs were based on the backbone of mir-1 with perfect complementarity between the guide and passenger strands, which favors loading onto AGO2 effector complexes. A shRNA library representing 83,256 unique synthetic hairpins was synthesized on four custom 22K Agilent microarrays, with up to six shRNAs per gene. Synthetic hairpin constructs were then amplified and cloned into pVALIUM20. The attB sequence allowed for targeted phiC31-mediated integration at genomic attP landing sites.

Constructs of the TRiP-3 and TRiP-4 collections deliver a short hairpin RNA that is more effective at knockdown than the long hairpin RNAs used in the first generation TRiP-1 and TRiP-2 construct collections.

The pVALIUM20-based constructs of the TRiP-3 collection give excellent knockdown in the soma and work well in the female germline, while the pVALIUM22-based constructs of the TRiP-4 collection give excellent knockdown in the germline and have little effect in the soma.

The second generation of shRNA-based RNAi constructs was developed because first generation, long dsRNA hairpin-based constructs are ineffective in gene knockdown during oogenesis.

In general, the phenotypes obtained using pVALIUM20-expressed shRNA constructs were stronger than those obtained with pVALIUM10-expressed long dsRNA ­hairpin constructs, and resembled genetic null mutations for the respective genes.

In compari­son to long­ hairpin constructs, shRNA constructs are expected to be advantageous as they give rise to only two siRNA species, the guide and passenger strands. However, off­target effects with shRNAs have been observed nonetheless, and verification of obtained phenotypes using independent shRNA constructs to the same gene is advised.

Many of the TRiP RNAi and sgRNA stocks generated at Harvard Medical School (HMS lines) and Tsinghua University (HMC lines), but not stocks generated at the National Institute of Genetics in Japan (HMJ lines), contain the sev²¹ allele as a result of introducing a mutated X chromosome from Bloomington stock 35781 (FBst0035781) or 32261 (FBst0032261) as the transgenes were being established in stock.