Expression of concatenated miR30-based shRNAs in a single transcript can promote efficient knockdown of at least three target genes. (a) Predicted secondary structure of the unprocessed miR30 transcript. Arrow depicts the location of the mature miRNA which is replaced with gene specific siRNA sequence in the expressed miR-shRNA. (b) Proposed structure of a transcript expressing multiple gene specific miR-shRNAs. (c) Subcloning scheme for creation of multi-miR-shRNA transcripts. Prom; any of the pol II promoters listed in Fig. 2a, attL1 + attL2; Gateway recombination sites, 5'miR + 3'miR; flanking sequence derived from human miR30. (d) Single CMV promoter-driven miR-shRNAs against arrestin 2 (Arr2), G beta 2 (Gβ2) and G-protein coupled receptor kinase 2 (Grk2) promote potent and specific knockdown when co-expressed with YFP-tagged cDNAs of their respective target genes in HEK293 cells. (e) Single transcripts expressing different combinations of double miR-shRNAs promote efficient dual target knockdown. (f) A single transcript expressing miR-shRNA against Arr2, Gβ2 and Grk2 retains potency against all three target genes.