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Post-transcriptional gene silencing has become the fastest and most frequently used approach to reduce gene function in cell culture. In an organism without an interferon response, such as Drosophila melanogaster, RNA interference (RNAi) can be evoked using long, double-stranded RNAs (1). In order to achieve a gene knockdown at a stage not amenable to dsRNA injection into early embryos, transgenic approaches have been used in Drosophila (2,3). Commonly, a 500 to 700 bp cDNA product is cloned as an inverted repeat with or without a spacer and expressed under the control of UAS-Gal4 sequences (4). To allow tissue-specific and/or temporally controlled expression of the RNAi transgene, the bimodular UAS-Gal4 system can be used, bypassing any potential toxicity of such transgenes.  Inverted repeats tend to be inherently difficult to clone in Escherichia coli due to recombination-mediated DNA repair that occurs during DNA replication (5). Lee and Carthew (6) designed pWIZ, a pUAST-based (4) fly transformation vector in which the two inverted repeats are separated by an intron of the white gene. After transcription, the intron is spliced out and the mRNA forms a hairpin in vivo. The cloning of inverted repeats into pWIZ is very difficult, even when sbcC− E. coli strains reducing recombination repair are used. A smaller shuttle vector in which the repeat/intron cassette is pre-assembled is easier to use, but requires at least 3 or 4 sub-cloning steps (7). Zhu and Stein used a strategy allowing directional cloning of inverted repeats in the germline UAS expression vector pUASp (8,9). pWIZ was also adapted for the GATEWAY in vitro recombineering system (Invitrogen, Carlsbad, CA, USA) (10). This elegant system, however, requires the purchase of expensive, special reagents.  In an attempt to simplify the generation of genomic RNAi constructs, we modified pWIZ to improve the cloning efficiency of dsRNA probes. The biggest problem during the cloning of inverted repeats is to obtain the inverted versus the direct orientation of the dsRNA target fragment during the second round of cloning. We therefore constructed pWIZdir by changing the polylinkers of pWIZ to allow the consecutive, directional insertion of a single PCR product using pairs of compatible restriction enzyme sets: SpeI/AvrII and NheI/XbaI adjacent to the white intron, and BglII and BamHI upstream and downstream, respectively (Figure 1). The directionality of the second cloning step greatly increases the frequency of obtaining correctly inverted repeats and obviates the need for the intermediate use of a shuttle plasmid. We have made eight dsRNAi constructs and obtained between 16% and 90% correctly inserted second-round cloning products, a significant improvement over previous attempts (not shown). All eight constructs were successfully used to generate transgenic fly lines. Examples of eye and wing phenotypes obtained in vivo are shown in Figure 2.    Figure 1  Schematic representation of pWIZdir and the directional cloning strategy      Figure 2  Examples of in vivo phenotypes generated by pWIZdir transgenes

Modified vectors for the two-step directional cloning of inverted repeats for RNA interference in Drosophila