Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster

Abstract

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.

Figure 1

Valium is an effective vector for transgenic RNAi. (a) Structure of Valium: Valium contains vermilion as the selectable marker; an attB sequence to allow for phiC31 targeted integration at genomic attP landing sites; two pentamers of UAS, one of which can be removed using the Cre-loxP system; the hsp70 TATA promoter; a multiple cloning site (MCS) that allows a single PCR product to be cloned in both orientations to generate the hairpin construct; the white intron, located between the two halves of the inverted DNA repeat, which has been shown to reduce toxicity in bacteria; and the ftz intron, followed by the SV40 poly(A) tail, to facilitate hairpin RNA expression, processing and export from the nucleus. We chose vermillion rather than mini-white because the exact gene dosage of white has been found to be important in behavioral studies. (b–g) Examples of hairpin-induced wing phenotypes in en-Gal4; UAS-hairpin flies. Note that the various phenotypes generated have no common features, indicating that there are no nonspecific patterning defects generated as a result of general overexpression of dsRNAs. Further, control hairpins do not show any unusual wing phenotypes (data not shown).

Figure 2

Parameters of in vivo RNAi using Valium. (a) Five classes of wing phenotypes were distinguished when a Notch hairpin was expressed using the wing margin C96-Gal4 driver. Class 1, wild type or a few bristles missing; class 2, margin bristles missing but no notches; class 3, moderate wing notching; class 4, extensive wing notching; class 5, most of the wing margin missing. (b) Phenotypes of C96-Gal4, Notch-hp2 flies. The phenotypes are stronger at 29 °C than at 25 °C and in males than in females; the two hairpins have similar expressivity. The effect of varying the number of UASs, the amounts of Gal4 and Dcr2 are shown using UAS-Notch hp2. More than 50 wings were scored in each experiment. Details of the construction of the various genotypes can be found in the Supplementary Note online.