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.

Valium is an effective vector for transgenic RNAi. (a) Structure of Valium: Valium contains vermilion as the selectable marker9; an attB sequence to allow for phiC31 targeted integration at genomic attP landing sites3; two pentamers of UAS, one of which can be removed using the Cre-loxP system10; 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 studies11. (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).