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Figure 5 Representative images of A) retinal organoids and B,C) zebrafish embryos transfected with mRNA lipoplexes. (A) Briefly, retinal organoids at day 22 of cultivation were incubated with (A2) 100% mCherry mRNA lipoplexes, (A3) 100% eGFP mRNA lipoplexes, (A4-A6) mingle-lipoplexes containing mCherry/eGFP mRNA at a ratio of 75/25%, 50/50%, 25/75% w/w or (A7-A9) mixtures of mCherry mRNA single-lipoplexes and eGFP mRNA single-lipoplexes at a ratio of 75/25%, 50/50%, and 25/75% w/w. The non-treated retinal organoids were used as control (A1). All the experiments were performed in triplicate (n = 9). At 24 h post-administration, retinal organoids were fixed by 2% PFA for images by microscopy. Scale bar: 100 µm. B) Zebrafish embryos of 48 h post fertilization were microinjected at the hindbrain ventricle with (B1) 100% mCherry mRNA lipoplexes, (B2) 100% eGFP mRNA lipoplexes, (B3-B5) mingle-lipoplexes containing mCherry/eGFP mRNA at a ratio of 75/25%, 50/50%, 25/75% w/w or (B6-B8,) mixtures of mCherry mRNA single-lipoplexes and eGFP mRNA single-lipoplexes at a ratio of 75/25%, 50/50%, and 25/75% w/w. At 24 h after injection, embryos were embedded in agarose and imaged using confocal microscope at 40x magnification (72 h post fertilization in total). C) Representative images of zebrafish embryos 24 h after injection (72 h post fertilization) at 10x magnification. (C1) schematic of a zebrafish embryo, (C2) mingle-lipoplexes containing mCherry/eGFP mRNA at a ratio of 50/50% (w/w and (C3) mixtures of mCherry mRNA single-lipoplexes and eGFP mRNA single-lipoplexes at a ratio of 50/50% w/w). Around 15–20 zebrafish embryos were injected for each sample. Scale bar: 100 µm. The total mRNA amount was kept constant at 1 µg/well for retinal organoids, and 0.2 ng/injection for zebrafish embryos.