Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila

Abstract

Whole-genome transgenic RNAi libraries permit systematic genetic screens in individual tissues of Drosophila . However, there is a high incidence of nonspecific phenotypes because of off-target effects. To minimize such effects, it is essential to obtain a deeper understanding of the specificity of action of RNAi. Here, in vivo assays are used to determine the minimum, contiguous nucleotide pairing required between an siRNA and a target mRNA to generate a phenotype. We observe that as few as 16 nucleotides of contiguous homology are sufficient to attenuate gene activity. This finding provides an explanation for the high incidence of off-target effects observed in RNAi-based genetic screens. Toward improving the efficacy of RNAi-induced phenotypes in vivo , we describe siRNA expression vectors that allow coexpression of one or more siRNAs with a fluorescent reporter gene in cultured cells or transgenic flies. This expression system makes use of the small intron from the ftz segmentation gene to provide efficient processing of synthetic siRNAs from a reporter transcript. These studies provide a foundation for the specific and effective use of gene silencing in transgenic Drosophila .

Essential role of grim-led programmed cell death for the establishment of corazonin-producing peptidergic nervous system during embryogenesis and metamorphosis in Drosophila melanogaster

“…Since microRNAs (miRNA)-based gene silencing has minimal cross-interference and more efficient knockdown effect (Chen et al, 2007; Haley et al, 2010; Ni et al, 2011), we employed this system. In the compound eyes, mi-grim and mi-rpr showed a complete rescue of GMR-grim and GMR - rpr -induced cell death, respectively, and these lines did not display cross-interference (Fig.…”

The let-7–Imp axis regulates ageing of the Drosophila testis stem-cell niche

“…Our search was focused on nucleotides 2–18 because previous studies suggested that pairing of the 5′ terminal nucleotide of the small RNA with its target is not required for efficient silencing. Furthermore, several mismatches between the 3′ end of the small RNA and the target site can be tolerated for target regulation 44,45 .…”

The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation

“…Using the Designer of Small Interfering RNA algorithm, we chose two sets of shRNA sequences for the TOP2 gene, with one targeting the protein-coding region and the other targeting the 3Ј UTR (21). In case of insufficient knockdown efficiency by a single shRNA, we employed two approaches to combine the effect of two shRNAs either from two separate plasmids or with tandem-linked shRNAs on a single plasmid (20). As described by Haley et al (20), the shRNA sequence is embedded in an ftz intron upstream of a GFP reporter gene, which allowed us to track the induction of the shRNA at the single cell level by fluorescence-activated cell sorting.…”

“…We adapted an intron-mediated shmiR expression system to generate an inducible plasmid-based shRNA system to simultaneously silence endogenous Top2 and express the WT or mutant Top2 (Fig. 6A) (20). Using the Designer of Small Interfering RNA algorithm, we chose two sets of shRNA sequences for the TOP2 gene, with one targeting the protein-coding region and the other targeting the 3Ј UTR (21).…”

“…Inducible Plasmid-based shRNA System for Cellular AssaysOur inducible plasmid-based shRNA system was adapted from an intron-mediated shmiR expression system by Haley et al (20), with modifications of restriction enzyme sites that facilitate switching between the constructs of shRNA sequences predicted from the Designer of Small Interfering RNA algorithm (Fig. 6A) (21).…”