Figure 3 of Semple-Rowland, Mol Vis 2007; 13:2001-2011.


Figure 3. Cellular activities of dual promoter lentivectors constructed by pairing GCAP292, mOP500, IRBP1783, and EF1α promoters in chicken retina

Retinas of embryos treated with either pFIN-GCAP292-GFP-IRBP1783-tdTomato (A-E), pFIN-IRBP1783-tdTomato-GCAP292-GFP (F-J), pFIN-IRBP1783-tdTomato-mOP500-GFP (K-O), pFIN-EF1α-tdTomato-GCAP292-GFP (P-U), or pFIN-EF1α-tdTomato-mOP500-GFP (V-BB). All whole mounts were photographed with the photoreceptors positioned nearest the objective. The color coded text in the lower right hand corner of each panel indicates the identities of the sources of the fluorescent signal(s) shown in the section. GFP and tdTom are green and tomato fluorescent proteins. The secondary fluorescent antibodies used to detect the rod transducin alpha antibody (RodTα) were either Alexa Fluor 488 (indicated by green text) or Alexa Fluor 594 (indicated by red text). All sections were counterstained with DAPI.

pFIN-GCAP292-GFP-IRBP1783-tdTomato: A: Retinal whole mounts showing the distribution of cell expressing GFP and tdTom. Cells co-expressing high levels of GFP and tdTomato are yellow. B-D: A representative section of retina treated with pFIN-GCAP292-GFP-IRBP1783-tdTomato. GFP (B) and tdTom (C) expression was limited to the photoreceptor layer in these retinas. D: The cells expressing similar levels of GFP and tdTom (arrow) were yellow when the images shown in B and C were merged. E: Retinal sections counterstained with RodTα and visualized with Alexa Fluor 488 (green) showed that tdTomato was not present in rod cells.

pFIN-IRBP1783-tdTomato-GCAP292-GFP: F: Retinal whole mount showing the distribution of cells expressing GFP and tdTom. G-I: A representative section of retina treated with pFIN-IRBP1783-tdTomato-GCAP292-GFP. tdTOM (G) and GFP (H) expression was limited to the photoreceptor layer in these retinas. I: The cells expressing similar levels of GFP and tdTom (arrow) were yellow when the images shown in G and H were merged. J: Retinal sections counterstained with RodTα and visualized with Alexa Fluor 594 (red) showed that GFP was not present in rod cells. pFIN-IRBP1783-tdTomato-mOP500-GFP: K: Retinal whole mount showing the distribution of cells expressing GFP and tdTom. L, M: Representative retinal sections showing expression of tdTOM (L) and GFP (M). Expression of both proteins was limited to the photoreceptor layer in these retinas. N: The section shown in panel M was immunostained with RodTα and visualized with Alexa Fluor 594 (red). O: Merge of images shown in M and N. Cells expressing GFP were rod cells (arrows).

pFIN-EF1α-tdTomato-GCAP292-GFP: P: Retinal whole mount showing the distribution of cells expressing GFP and tdTom. Photographs were taken so that the photoreceptor cell layer was in focus. Cells expressing tdTomato were present in the photoreceptor layer and in the inner layers of the retina (cells appearing out of focus are located in the inner retina). Q-S: Representative retinal sections showing expression of tdTOM and GFP. Expression of GFP was limited to the photoreceptor layer (Q) while tdTomato was present in all cell layers (Q, S). Cells co-expressing these proteins were located in the photoreceptor cell layer (R). T, U: Section showing expression of tdTomato (T) that was counterstained with RodTα (U). Examination of several sections revealed that very few rod cells contained tdTomato (U, arrow).

pFIN-EF1α-tdTomato-mOP500-GFP: V: Retinal whole mount showing the distribution of cells expressing GFP and tdTom. Photographs were taken so that the photoreceptor cell layer was in focus. Cells expressing GFP and tdTomato were present in the photoreceptor layer and in the inner layers of the retina (cells appearing out of focus are located in the inner retina). W-Y: Representative retinal sections showing expression of tdTOM and GFP. GFP and tdTomato was present in all cell layers (W, X). Z, AA. GFP was localized to rod cells in the photoreceptor cell layer (Z). Immunostaining with RodTα (AA) confirmed that GFP was in rod cells. In this image, a GFP-positive transduced Müller cell (arrow) was observed. The presence of GFP in the outer limiting membrane (OLM dashed line) is consistent with this observation. BB: Very few of the rod cells stained with RodTα were also positive for tdTomato. Scale bars in A, F, K, P and V equal 50 μm. Scale bars in B, G, L, T, and Z equal 10 μm. The images shown in C-E, H-J, M-O, U, AA and BB were photographed at the same magnification as those shown in B, G, L, T, and Z. Scale bars in Q and W equal 20 μm. The images shown in R, S, X, and Y were photographed at the same magnification as those shown in Q and W. Abbreviations: OS represents outer segments; ONL represents outer nuclear layer; OPL represents outer plexiform layer; INL represents inner nuclear layer; OLM represents outer limiting membrane.

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Semple-Rowland, Mol Vis 2007; 13:2001-2011 <http://www.molvis.org/molvis/v13/a225/>
©2007 Molecular Vision <http://www.molvis.org/molvis/>
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