Fig. 5: Development of CLDN5 MiniPromoters for endothelial cells of the blood–retina barrier.

A Bioinformatic design of first-generation Ple32, and redesign to produce second- and third-generation Ple261, Ple338, Ple339, and Ple340. ENCODE segments that potentially regulate the expression of the CLDN5 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 4 intravenous injection of Ple32-EmGFP, harvested 1 week later, led to robust expression in the blood vessels, as determined by morphology. Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. S1A.) C Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, and visualized by whole mount retina, led to robust expression in the blood vessels, as determined by morphology. Scale bar, 100 µm (For smCBA see Fig. S1F.) D Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in CD31-positive cells, and showed significantly higher (~2×) expression compared to smCBA (p < 0.01). (For smCBA see Fig. S1F). E Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, led to robust expression in the endothelial cells of the blood–retina barrier, as indicated by co-staining with the endothelial cell marker CD31. Scale bar, 20 µm. (For smCBA see Fig. S1F.) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-CD31; yellow, merge.