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Figure 1. Cellular activities of interphotoreceptor retinoid binding protein promoter (IRBP156), Xenopus opsin promoter (XOPS), neural retina leucine zipper (NRLL), and rhodopsin kinase (RK) promoters in chicken retina. Lentiviral vectors were injected into the ventricles of chicken embryos (embryonic day 2 –E2) in ovo. The viruses injected were as follows: A-B pFIN-IRBP156-tdTOM; C-D pFIN-XOPS-tdTOM; E-F pFIN-NRLL-tdTOM; G-H pFIN-RK-GFP-WPRE. The retinas of the injected embryos were harvested on E19–20 and the cells expressing the fluorescent reporter proteins were identified using native fluorescent and immunofluorescent microscopy. In selecting the representative images shown in this and subsequent figures, our goal was to document all of the cell types and the variability in expression levels of the reporter proteins observed in transduced cell populations. The sections shown in B, D, F, and H were immunostained with a rod transducin polyclonal antibody that was visualized using either goat anti-rabbit Alexa Fluor 488 (B, D, F) or 594 (H) secondary antibody. Arrows indicate inner retinal cells (A) or rod photoreceptors (B, H). All sections were counterstained with DAPI. All scale bars shown equal 50 µm. Abbreviations: ONL represents outer nuclear layer; INL represents inner nuclear layer.
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Figure 2. Cellular activities of cluster differentiation (CD)44, vimentin (VIM), and glial fibrillary acidic protein (GFAP) promoters in chicken retina. Lentiviral vectors were injected into the ventricles of chicken embryos (embryonic day 2–E2) in ovo. The retinas of the injected embryos were harvested on E19–20 and the cells expressing the fluorescent reporter proteins were identified using native fluorescent and immunofluorescent microscopy. The viruses injected were as follows: A-I pFIN-CD44-GFP-WPRE; J-Q pFM-VIM-GW; R-V pFM-GFAP-GW. All sections were counterstained with DAPI, and all scale bars shown equal 50 µm. Abbreviations are as follows: ONL represents outer nuclear layer; INL represents inner nuclear layer; IPL represents inner plexiform layer; GCL represents ganglion cell layer. CD44: A: Photograph of whole mount of retina that had been treated with pFIN-CD44-GFP-WPRE. Clusters of GFP-positive photoreceptors (arrows) were detected across the surface of the whole mount. B: This image was produced by re-photographing the boxed region shown in A using a focal plane just below that used to obtain the image shown in A. Horizontal cells (asterisks) were the predominant GFP-positive cell type observed in this focal plane. C-I These images represent sections of retinas showing the cell types (arrows) in which the CD44-GFP transgene was active (C photoreceptors, D horizontal cells, E amacrine cells, F-H Müller cells, I ganglion cells). Section shown in F was counterstained with an antibody against chicken carbonic anhydrase II (CAII), a marker for Müller cells (G). The merged image (H) shows that the GFP-positive cells also expressed carbonic anhydrase II. VIM: J, K Photographs of a whole mount of a retina treated with pFM-VIM-GW and viewed from the photoreceptor side of the whole mount. J Numerous GFP-positive horizontal cells were detected in the transduced retina (arrow). K Enlargement of the region in image J (box) that contains GFP-positive horizontal cells (arrow). L This image was produced by re-photographing the boxed region shown in K using a focal plane just below that shown in K. Müller cell bodies are the predominant cell type observed in this image plane (asterisk). The horizontal cell indicated in J, K, and L by the arrow is the same cell. M,N Images of sections of the retinal whole mount shown in J and K. GFP-positive horizontal (M, arrow), Müller (M, asterisk), and photoreceptor (N, ONL) cells were detected in several sections. O-Q A section containing GFP-positive cells located in the INL (O, arrow) was counterstained with an antibody against chicken carbonic anhydrase II (P). The merged image (Q) shows that the GFP-positive cells also expressed carbonic anhydrase II. GFAP: R, S Images of a whole mount of a 5-week old GUCY1*B chicken retina that had been treated with pFM-GFAP-GW on E2 and photographed from either the photoreceptor (R) or the vitread (S) side of the whole mount. The pattern of GFP localization observed in these whole mounts suggested that the cells expressing the GFAP-GFP transgene were Müller cells. T-V Sections of the transduced retinas showed that the cell bodies of the GFP-positive cells observed in R and S were located in the INL (T). Immunostaining of these sections with an antibody against chicken carbonic anhydrase II (U) revealed that the GFP-positive cells also expressed carbonic anhydrase II (V).
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Figure 3. Expression of photoreceptor promoter-driven fluorescent proteins in retinas transduced with mixtures of two lentiviruses. Lentiviral vectors carrying transgenes comprised of various photoreceptor promoters driving expression of GFP or tdTOM fluorescent proteins were mixed in equal volumes and injected into the developing neural tubes of chicken embryos (embryonic day 2 –E2) in ovo. The injected virus mixtures were as follows: A-E: pFIN-GCAP292-GFP (2.2×1010 vector genomes/µl) and pFIN-IRBP1783-tdTOM (1.6×1010 vector genomes/µl); F-J: pFIN-RK-GFP-WPRE (1.2×1010 vector genomes/µl) and pFIN-IRBP1783-tdTOM (1.6×1010 vector genomes/µl); K-O: pFIN-RK-GFP-WPRE (1.2×1010 vector genomes/µl) and pFIN-IRBP156-tdTOM (5.6×1010 vector genomes/µl); P-T: pFIN-GCAP292-GFP (2.2×1010 vector genomes/µl) and pFIN-XOPS-tdTOM (1.2×109 vector genomes/µl). We have previously shown that the GCAP292 and IRBP1783 promoters are active in cone cells [18]. RK and IRBP156 are active in both rod and cone cells and XOPS is only active in rod cells (Figure 1). For each image series, the transduced retina was photographed from the photoreceptor side of the whole mount using GFP (A, F, K, P) and CHER (B, G, L, Q) filters. These images were then merged to identify cells expressing both reporter proteins (C, H, M, R). The merged images were analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The results of these analyses are expressed as the percent of the transduced area in the image (pixels) containing co-localized GFP and tdTOM (yellow bar) or GFP (green bar) or tdTOM (red bar) fluorescence alone (D, I, N, S). The images shown in E, J, O, and T were extracted from the merged images shown in C, H, M, and R and show only those areas of the merged image in which GFP was co-localized with tdTOM. The scale bar shown in A is applicable to all images and equals 50 µm.
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Figure 4. Expression of elongation factor 1a (EF1a) promoter-driven fluorescent proteins in retina and brain transduced with a mixture of two lentiviruses. Equal volumes of two lentiviral vectors, pFIN-EF1a-GFP-WPRE (2.4×1010 vector genomes/µl) and pFIN-EF1a-CHER-WPRE (4.6×109 vector genomes/µl), were mixed and injected into the developing neural tubes of E2 chicken embryos in ovo. The retinas and brains of the injected embryos were harvested on E19-E20 and the cells expressing GFP and CHER were identified using fluorescent microscopy. A, B These retinal whole mounts, viewed from the photoreceptor side, show the distribution of retinal cells expressing GFP and/or CHER fluorescent protein(s). The scale bars in A and B equal 2000 and 50 µm, respectively. C-D and E-H: Sections of whole mounts shown in A and B show that the EF1a promoter is active in cells distributed throughout the neural retina. C: This retinal section, which contains very few transduced cells expressing both GFP and CHER, was typical of most regions of the transduced retinas. D: The image shown in C was analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The results of these analyses are expressed as the percent of the transduced area in the image (pixels) containing co-localized GFP and CHER (yellow bar) or GFP (green bar) or CHER (red bar) fluorescence alone. E-G: Image of retinal section showing green (E), red (F) and merged (G) channels that contain several cells expressing both GFP and CHER. H: The image shown in E was analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The results of this analysis are expressed as the percent of the transduced area in the image (pixels) containing co-localized GFP and CHER (yellow bar) or GFP (green bar) or CHER (red bar) fluorescence alone. The scale bars in C and D equal 50 µm. I: Thionin stained sagittal section of E20 chicken brain. Scale bar equals 1000 µm. J-L: Fluorescent images of GFP and CHER expression in optic tectum (the region shown in F-F’’ corresponds to the boxed region in I). The brain sections were stained with a chicken anti-GFP antibody to enhance visualization of the GFP expressing cells. Tectal layers are numbered according to Cajal [43]. Scale bar in J equals 100 µm. Retinal and brain sections shown in C, D-G and J-L were counterstained with DAPI.
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Figure 5. Expression characteristics of dual promoter vectors constructed using rhodopsin kinase (RK) and interphotorecepter binding protein (IRBP)156 promoters. pFIN-RK-GFP-IRBP156-CHER-WPRE (3.3×107 vector genomes/µl; A-E and F-J), pFIN-IRBP156-CHER-RK-GFP-WPRE (1.5×109 vector genomes/µl; K-O and P-T), pFIN-RK-GFP-RK-CHER-WPRE (1.85×107 vector genomes/µl; U-Y and Z-DD), or pFIN-IRBP156-CHER-IRBP156-GFP-WPRE (1.7×109 vector genomes/µl; EE-II and JJ-NN) lentivirus was injected into the developing neural tubes of E2 chicken embryos in ovo. A minimum of four retinal whole mounts were examined for each virus. Retinal regions shown in the figure were selected to illustrate the range of transgene expression characteristics observed in infected cells. Each region was photographed twice using the exposure duration shown in the lower left of each panel and filters appropriate for detection of CHER or GFP. Each row in the figure shows one selected region. The merged images (C, H, M, R, W, BB, GG, LL) were analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The results of these analyses are expressed as the percent of the transduced area in the image (pixels) containing co-localized GFP and CHER (yellow bar) or GFP (green bar) or CHER (red bar) fluorescence alone. (D, I, N, S, X, CC, HH, MM). The images shown in E, J, O, T, Y, DD, II, NN were extracted from the merged images (C-LL) and show only those areas of the merged image in which GFP was co-localized with CHER. The scale bar shown in A is applicable to all images and equals 50 µm.
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Figure 6. Cellular specificity of the pFIN-IRBP156-CHER-RK-GFP-WPRE dual-promoter vector. pFIN-IRBP156-CHER-RK-GFP-WPRE (1.5×109 vector genomes/µl) lentivirus was injected into the developing neural tubes of E2 chicken embryos in ovo. The retinas of the injected embryos were harvested on E19–20, examined as whole mounts using native fluorescent, and sectioned (10 µm). A-C: A representative section showing the extent of photoreceptor infection. The merged image indicates that nearly all cells are co-expressing the two fluorescent reporter proteins, CHER and GFP. D-F: Close up of transduced photoreceptor layer in region containing INL cells expressing the viral transgene (arrow in D). All sections were counterstained with DAPI. All scale bars shown equal 50 µm. Abbreviations: ONL represents outer nuclear layer; INL represents inner nuclear layer; IPL represents inner plexiform layer.
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Figure 7. The effects of internal insulators on the expression of the RK-GFP and IRBP156-CHER transgenes carried by the pFIN-RK-GFP-IRBP156-CHER-WPRE vector. Examination of the expression of GFP and CHER in retinas transduced with pFIN-RK-GFP-HS4(2×250)F-IRBP156-CHER-WPRE (3.3×108 vector genomes/µl; A-J) or pFIN-RK-GFP-HS4(1.2)F-IRBP156-CHER (1.2×108 vector genomes/µl; K-T). Each lentivirus was injected into the developing neural tubes of E2 chicken embryos in ovo. The retinal whole mounts were photographed twice using the exposure duration shown in lower left of each panel and filters appropriate for detection of GFP or CHER. The GFP and CHER images were merged (C, H, M, R) and analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The results of these analyses are expressed as the percent of the transduced area in the image (pixels) containing co-localized GFP and CHER (yellow bar) or GFP (green bar) or CHER (red bar) fluorescence alone D, I, N, S. The images shown in E, J, O, T were derived from the merged images (C, H, M, R) and show only those areas of the merged image in which GFP and CHER were co-localized. The scale bar shown in A is applicable to all images and equals 50 µm.
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Figure 8. Expression characteristics of dual promoter vectors constructed using murine opsin promoter (MOPS) and Xenopus opsin promoter (XOPS) promoters. pFIN-XOPS-tdTOM-MOPS-GFP (1.6×108 vector genomes/µl; A-J) or pFIN-MOPS-GFP-XOPS-tdTOM-WPRE (4.4×108 vector genomes/µl; K-T) lentivirus was injected into the developing neural tubes of E2 chicken embryos in ovo. Retinal whole mounts (one retina per horizontal row) were photographed twice using the exposure duration shown in lower left of each panel and filters appropriate for detection of tdTOM or GFP. The merged images (C, H, M, R) were analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The relative percent area (pixels) of each image containing GFP (green bar) or tdTOM (red bar) fluorescence alone or both GFP and tdTOM (yellow bar) is shown in panels D, I, N, S. The images shown in E, J, O, T were derived from the merged images (C, H, M, R) and show only those areas of the merged image in which GFP was co-localized with CHER. The scale bar shown in A is applicable to all images and equals 50 µm.
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Figure 9. Expression characteristics of a dual promoter vector constructed using cluster differentiation (CD)44 and vimentin (VIM) promoters. pFIN-CD44-CHER-VIM-GFP-WPRE lentivirus was injected into the developing neural tubes of E2 chicken embryos in ovo. Retinal whole mounts were photographed twice using filters appropriate for detection of GFP (A) or CHER (B) and the exposure times (ms) shown in the lower left of the images. The GFP and CHER images were merged (C) and analyzed using the co-localization module of the Zeiss AxioVision Image Suite. The relative percent area (pixels) of the merged image containing GFP (green bar) or CHER (red bar) fluorescence alone or both GFP and CHER (yellow bar) is shown in panel D. Sections of the transduced retinas revealed that GFP expression was largely restricted to Müller cells (arrow, E). A few GFP-positive horizontal cells were detected in these retinas but their numbers were reduced relative to the numbers of these cells that were present in retinas transduced with pFM-VIM-GW (Figure 2J-N). Scale bars shown in A and E equal 50 µm.
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