Lentiviral Delivery of Proteins Using 2A Peptides Figure 4. Evaluation of the expression characteristics of the EF1-GFP-2A-GC1 transgene in transiently transfected HEK 293FT cells. (A) Diagram of the EF1-GFP-2A-GC1 transgene. (B–E and F–I) Representative images of transiently transfected HEK 293FT cell cultures expressing EF1-GFP-2A-GC1 imaged 48 h post-transfection. Scale bars shown in B and F equal 100 and 50 mm, respectively. (J–M) Western blot analyses of HEK 293FT cells transiently transfected with pFIN-EF1-GFP-2A-GC1-WPRE (n = 3) probed for GC1 (J), GFP (K), 2A (L), and GAPDH (M). Non-transfected HEK 293FT cells (NT), HEK 293FT cells transfected with pFIN-EF1-GC1-IRES-GFP (IRES), and HEK 293FT cells transfected with pFIN-EF1-GFP-WPRE served as controls. GAPDH served as a loading control. (N) The cleavage efficiency of GFP-2A-GC1 was estimated by expressing the intensities of the cleaved GFP-2A (K) protein bands as a percentage of total GFP immunoreactivity (intensity of the cleaved plus uncleaved protein bands). The estimate is the mean6SD of three samples. doi:10.1371/journal.pone.0020553.g004 chickens failed to respond to the stimuli (Video S2). All of the treated embryos that hatched produced OKN responses (Figure 5B; Video S3). A repeated measures ANOVA was used to examine the effects of stimulus type on the magnitude of the OKN responses over time. The results indicated that the response magnitude significantly decreased over the course of the 5-week test period (F(5,60) = 5.0; p = 0.0007), in the absence of a main effect of stimulus type. At the end of the 5-week testing period, we analyzed whole mounts of treated animal’s retinas to obtain an estimate of the efficiency of our viral treatment. Our viral delivery method leads to infection of progenitor cells in the developing neural tube, some of which generate the neural retina. Examples of the viral transduction patterns produced in the retinas of our treated animals are shown in Figure 5 (C,D). Unlike the transduction pattern produced following subretinal delivery of viral vectors that is characterized by one large, contiguous population of infected cells, the transduction pattern produced following neural tube delivery of viral vectors is characterized by numerous scattered groups of infected cells that are distributed across the entire retina. The estimates of the total area transduced by the virus in the retinas shown in Figure 5 were 7% and 16%. These retinas belonged to the animal in the treated group whose OKN responses were assigned a rating of 3 throughout the entire 5-week test PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20553