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. 2021 Feb 2;28(6):351–372. doi: 10.1038/s41434-021-00227-z

Fig. 7. Development of PAX6 MiniPromoters.

Fig. 7

A Bioinformatic design of first-generation Ple255 and Ple259, which were combined to produce the second-generation redesigns Ple328 and Ple329, also bioinformatic design of SIMO element Ple330 and base pair modified Ple331. ENCODE segments that potentially regulate the expression of the PAX6 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 0 intravenous injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the ganglion cell layer (GCL), and the inner nuclear layer (INL). Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. S1A). C Postnatal day 4 intravenous injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the GCL, INL, and processes that span the retina. Scale bar, 100 µm. (For smCBA see Fig. S1A.) D Adult subretinal injection of Ple331-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in RBPMS-positive ganglion cells, and showed significantly lower (~30%) ganglion cell expression when compared to smCBA (p < 0.01). (For smCBA see Fig. S1C.) E Adult subretinal injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression in the PAX6-positive cells, ganglion, amacrine, horizontal, and Müller glia, as indicated by co-staining with anti-PAX6. Scale bar, 20 µm. (For smCBA see Fig. S1C.) EmGFP emerald green fluorescent protein, 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-Pax6; yellow, merge.