RNAi Library

The library comprises 22,247 transgenic Drosophila strains, each containing an inducible UAS-RNAi construct against a single protein coding gene. 12,251 genes, or 88.2% of the Drosophila genome, are represented in this collection. All insertions have been molecularly validated, and a sample also functionally validated. We estimate that >80% of the lines provide potent and gene-specific silencing.

Figure 1: Transgenic RNAi in Drosophila. The generic GAL4/UAS system is used to drive the expression of a hairpin RNA (hpRNAs). These double-stranded RNAs are processed by Dicer into siRNAs which direct sequence-specific degradation of the target mRNA.

The library was constructed by cloning short gene fragments (300-400bp) as inverted repeats (IRs) in the antisense-sense orientation into a modified pUAST vector pMF3 (see the map in the protocols) with 10 copies of UAS sites.
To amplify 300-400bp gene fragments primers were designed to amplify from every predicted protein-coding gene in Release 4.3 of the Drosophila genome sequence. 15,072 PCR products were successfully amplified and cloned as IRs into pMF3, representing 13,344 genes or 97% of Drosophila genome.

Figure 2: RNAi phenotypes. (A). Control with GAL4 driver only. (B). GAL4 driver + UAS-eyRNAi, targeting the eyeless gene. The eye is missing, as in the eyeless mutant. (C). Wing hairs in a wild-type fly all point in the same direction. (D). GAL4 driver + UAS-fmiRNAi, targeting flamingo, a gene required for planar cell polarity. The wing hairs are misorientated, as in the flamingo mutant.