Lentiviral Delivery of Proteins Using 2A Peptides Figure 3. Expression characteristics of bicistronic lentiviruses in chicken retina. Lentiviruses were injected and retinal whole mounts and sections were imaged as described in the methods. (A–C) Whole mount and (D–F) representative section of a retina treated with pFIN-EF1-GFP-EF1- mCher-WPRE virus show robust expression of the downstream reporter (mCher). The upstream reporter (GFP) was either weakly expressed (A) or not detected (D). (G–I) Whole mount and (J–L) representative section of a retina treated with pFIN-EF1-GFP-WPRE-EF1-mCher-WPRE virus revealed that both reporter proteins were expressed in the treated retina but that only one of the reporter proteins could be detected in the majority of cells expressing the transgene. (M-O) Whole mount and (P–R) representative section of a retina treated with pFIN-EF1-GFP-2A-mCher-WPRE show robust co-expression of both reporter proteins in transduced cells. Scale bars in A and D = 50 mm. (S–V) Western blot analyses of the retinas of four chickens treated with pFIN-EF1-GFP-2A-mCher(H)-WPRE virus. Four replicate blots containing proteins extracted from the retinas were probed with antibodies to either hemagglutinin (S), GFP (T), 2A (U), or GAPDH (V). (W) The cleavage efficiency of GFP-2A-mCherH was estimated by expressing the intensities of the cleaved GFP-2A (T) or mCherH (S) protein bands as a percentage of total GFP or mCherH immunoreactivity (intensity of the cleaved plus uncleaved protein bands), respectively. Each estimate is the mean6SD of three samples. doi:10.1371/journal.pone.0020553.g003 GC1 was localized primarily to the cell membranes (Figure 4G,H) while GFP filled the cell bodies (Figure 4F,H), locations consistent with the expected subcellular trafficking of these proteins. Western blot analyses of cells transfected with pFIN-EF1-GFP- 2A-GC1-WPRE showed that the cell lysates contained cleaved GC1 (Figure 4J) and GFP-2A (Figure 4K,L). Uncleaved GFP-2A- GC1 peptide was detected but only when the blots probed with the GFP antibody were highly overexposed. Quantification of the cleavage efficiency of the GFP-2A-GC1 peptide revealed that it was 95.3%61.3 (GFP, M6SD; n = 3) (Figure 4N). Evaluation of the Therapeutic Potential of pFIN-EF1-GFP-2A-GC1-WPRE In our final experiment, we set out to determine if the integrated EF1-GFP-2A-GC1 transgene would produce sufficient levels of GC1 to restore function to the photoreceptors of GUCY1*B chickens. To be effective, the 2A fusion peptide expressed in photoreceptors needed to be cleaved efficiently and the resulting GC1 enzyme had to be properly trafficked to the outer segments of the photoreceptors. Stage 8–10 GUCY1*B embryos were injected with pFIN-EF1-GFP-2A-GC1-WPRE, hatched, and tested weekly for the presence of the optokinetic visual response (OKN) until they reached 5 weeks-of-age. The OKN responses of 7 treated animals were evaluated once a week for five weeks by holding the animals in the center of rotating platform that supported high contrast, visual stimuli consisting of vertical square wave gratings with spatial frequencies of 0.065 or 0.26 cycles?degree21 that corresponded to bar widths of 5 or 1.25 cm, respectively (Figure 5A). These responses were compared to those obtained from wild-type and untreated GUCY1*B animals. OKN responses are driven primarily by the peripheral regions of the retina [29,30], and unlike mammalian OKN responses, these responses in chickens are monocularly driven, the responses to clock-wise and counter-clockwise rotation of the visual stimuli being driven by the left and right eyes, respectively [31]. The OKN responses of the wild-type chickens were robust, characterized by well-defined lateral head movements synchronized with the speed of rotation of the visual stimulus (Video S1), while the untreated GUCY1*B PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20553