A genome-scale shRNA resource for transgenic RNAi in Drosophila

Abstract

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

Figure 1

Design of shRNA constructs and phenotypes of shRNA-mediated gene silencing. (a) Structure of the Drosophila miR-1 and shRNA hairpins (miR-1 nucleotides replaced by the sequence of interest are indicated by N). (b) Phase contrast images showing ovary phenotypes associated with knockdown of bag-of-marbles (shRNA to bam, labeled shRNA-bam) and ovarian tumor (shRNA-otu) in MTD-Gal4/UAS-shRNA females (using Valium20). DAPI images show single tumorous egg chamber and wild-type egg chamber. Scale bars, 500 μm (phase contrast) and 200 μm (DAPI). (c) Dark field images of the cuticle of wild-type embryo and embryos derived from MTD-Gal4/UAS-shRNA females. Scale bars, 100 μm. (d) Knockdown of Notch in the wing using C96-Gal4/UAS-shRNA-N. Scale bars, 400 μm. (e) Knockdown of white using GMR-Gal4. In the labels, hp stands for hairpin. Scale bars, 100 μm.

Figure 2

Analysis of the piRNA pathway during oogenesis. (a) Immunofluorescence staining of early egg chambers showing depletion of the indicated piRNA pathway components using specific antibodies (green) upon shRNA expression via MTD-Gal4 (using Valium22). DNA was visualized with DAPI (blue). Black and white images are of the antibody staining only. Scale bars, 20 μm. (b) Fertility rates of females in which the indicated genes were knocked down with shRNAs in the germline via MTD-Gal4 (using Valium22). For each knockdown 300–500 eggs were counted. (c) Fold changes in steady-state RNA levels of the transposable elements HeT-A and blood in comparison to the germline-specific nanos transcript upon knockdown of the indicated genes via shRNAs. The data were compared to a control sample in which the white gene was knocked down (rp49 transcript levels were used for normalization). Data are averages of three independent biological replicates; error bars, s.d. (d) Immunofluorescence staining of early egg chambers with an antibody to the I-element ORF1p. Left two images are of flies expressing shRNA-spn-E with MTD-Gal4 (using Valium22); right two images are of wild-type flies. DNA was visualized with DAPI (blue).