transgene (Figure 2I). Relative levels of mCherH produced from all three transgenes were similar (Figure 2B, F, J). Merged GFP and mCherH images (Figure 2C, G, K) were examined to determine if the levels of expression of these proteins was uniform across infected cells. Cells infected with pFIN-EF1-GFP-EF1- mCherH-WPRE virus contained high levels of mCherH and very little to no detectable GFP (Figure 2C), while those infected with pFIN-EF1-GFP-2A-mCherH-WPRE virus contained high levels of both proteins. The few cells infected with pFIN-EF1-GFP-EF1- mCherH-WPRE virus that did contain trace amounts of GFP did not appear to express mCherH. Expression of GFP and mCherH was the least uniform in cultures transduced with pFIN-EF1-GFP- W-EF1-mCherH-WPRE virus (Figure 2G). The majority of the cells in these cultures expressed either GFP or mCherH; however, a subpopulation of the cells was present that co-expressed GFP and mCherH as evidenced by their yellow hue in the merged image (Figure 2G, arrows). The expression of each transgene was also analyzed using western blot (Figure 2M–P). Blots were prepared in quadruplicate and were probed with antibodies against either the HA tag (Figure 2M), GFP (Figure 2N), 2A (Figure 2O) or GAPDH (Figure 2P). The relative amounts of GFP and mCherH produced from each transgene were determined using a scanning densitom- eter and the values were normalized to the highest value obtained for each protein, respectively (Figure 2Q). Uncleaved GFP-2A- mCherH was not included in the determination of the amount of GFP and mCherH produced in cells expressing the EF1-GFP-2A- mCherH transgene. An analyses of variance revealed significant differences between transgenes in terms of the amount of GFP expressed (F(2,6) = 315.13, p,0.001); post hoc analyses using Tukey tests showed that the level of GFP produced by EF1-GFP- W-EF1-mCherH (M = 0.25, SD = 0.05) was significantly lower than that produced by EF1-GFP-2A-mCherH (M = 0.95, SD = 0.06). The levels of GFP produced by EF1-GFP-EF1- mCherH were not detectable on western blots but very low levels of this protein could be detected in a small minority of the transduced cultured cells (Figure 2A). No differences were observed in the levels of mCherH produced by the three transgenes (F(2,6) = 4.22, p = 0.072). Estimates of the cleavage efficiency of GFP-2A-mCherH were calculated by expressing the intensities of the cleaved mCherH (Figure 2M) or GFP-2A (Figure 2N) protein bands as a percentage of total HA or GFP immunoreactivity, respectively. The estimates produced from these calculations were 96%60.6 (GFP, M6SD; n = 3) and 62%62.3 (HA, M6SD; n = 3) (Figure 2R). Expression of the Packaged Viral Bicistronic Transgenes in Chicken Retina In this series of experiments, the three bicistronic lentiviruses were injected into chicken embryos to determine if their expression characteristics in neural retina were similar to those observed in HEK 293FT cells. Retinal whole mounts and cryosections were examined using fluorescent microscopy to determine if GFP and mCherH could be detected in infected cells and if the relative amounts of these proteins were uniform among these cells. Examination of whole mounts (Figure 3A–C) and sections (Figure 3D–F) of retinas transduced with pFIN-EF1-GFP-EF1- mCher-WPRE revealed that the relative expression levels of mCherH were much higher than GFP in these retinas; the few cells that expressed detectable levels of GFP did not appear to be co-expressing mCher. Both reporter proteins were detected in retinas transduced with pFIN-EF1-GFP-WPRE-EF1-mCher- WPRE; however, the vast majority of the cells expressing this bicistronic transgene were positive for either GFP or mCher Lentiviral Delivery of Proteins Using 2A Peptides (Figure 3G–I). We were able to identify scattered groups of cells in retinal sections that expressed both reporter proteins, but they were rare and the relative amounts of each protein in these cells were highly variable (Figure 3J–L). Finally, examination of retinas transduced with the pFIN-EF1-GFP-2A-mCher-WPRE virus showed that both of the reporter proteins encoded by this virus were robustly expressed in these retinas (Figure 3M–R) and that the relative amounts of these proteins in infected cells were similar. Together, these results indicate that the expression characteristics of these three viruses are similar in HEK 293FT and chicken neural retinal cells. The variability in the percent of the total area of the retinas of embryos transduced by the bicistronic viruses precluded the use of western blot analyses of homogenates of these retinas to determine the relative expression levels of GFP and mCherH generated from each of the viral transgenes. However, we were able to examine the cleavage efficiency of the GFP-2A-mCherH peptide within individual retinas transduced with pFIN-EF1a-GFP-2A-mCherH- WPRE virus using western blot (Figure 3S–V). Blots containing proteins isolated from the retinas of four chickens that had been treated with pFIN-EF1a-GFP-2A-mCherH-WPRE virus and one untreated control retina (WT) were probed with antibodies recognizing either the HA tag (Figure 3S), GFP (Figure 3T), 2A (Figure 3U) or GAPDH (Figure 3V). Examination of the HA tag and GFP staining patterns suggested that cleavage of the GFP-2A- mCherH peptides in infected retinal cells was efficient, results that were corroborated by the 2A staining pattern (Figure 3U). Estimates of the cleavage efficiency of GFP-2A-mCherH were calculated by expressing the intensities of the cleaved mCherH (Figure 3S) or GFP-2A (Figure 3T) protein bands as a percentage of total HA or GFP immunoreactivity, respectively (Figure 3W). The estimates produced from these calculations were 99%60.4 (GFP, M6SD; n = 4) and 91%62.6 (HA, M6SD; n = 4). These data show that our bicistronic transgene carrying a 2A cleavage sequence can be properly transcribed and translated in retinal cells in vivo. Expression of 2A Bicistronic Transgenes Encoding Guanylate Cyclase-1 in Transiently Transfected HEK 293FT Cell Cultures Prompted by the excellent expression characteristics exhibited by the 2A containing reporter lentiviruses in vitro and in vivo, we constructed a therapeutic 2A vector encoding guanylate cyclase -1 (GC1) and examined its expression in transiently transfected HEK 293FT cells. The transgene we constructed encoded GFP, which was used to identify transfected or transduced cells, and the gene encoding GC1that was fused in frame to the 2A peptide downstream of GFP (Figure 4A). GC1 is a transmembrane protein that is normally trafficked to the photoreceptor outer segments where it plays a critical role in recovery of these cells from light stimulation [12]. We have previously shown that delivery of bovine GC1 to the photoreceptors of the GUCY1*B chicken model of LCA1 in the context of an IRES-containing lentiviral transgene (pTYF-EF1-GC1-IRES-GFP) temporarily reversed blindness in these animals [15]. In this experiment, we evaluated the expression of EF1-GFP-2A-GC1 in transiently transfected 293FT cells using fluorescent microscopy and western blot. Examination of HEK 293FT cell cultures transfected with pFIN-EF1-GFP-2A-GC1-WPRE DNA (Figure 4B–I) revealed that all transfected cells expressed GFP and GC1. In normal photoreceptor cells, GC1 is localized to the membranes of the outer segments. Thus, we expected that the GC1 produced from the GFP-2A-GC1 peptide would localize to the membranes of the transfected cells, which was found to be the case. Immunostained PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20553