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.