Fig. 6: In vivo gene editing with Cre-UCNPs followed by functional analyses of the edited cells by optogenetics. | Nature Communications

Fig. 6: In vivo gene editing with Cre-UCNPs followed by functional analyses of the edited cells by optogenetics.

From: Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications

Fig. 6: In vivo gene editing with Cre-UCNPs followed by functional analyses of the edited cells by optogenetics.The alternative text for this image may have been generated using AI.

a Schematic representation of the in vivo setup. AAV5.dflox.ChR2-YFP was injected into the VTA together with Cre-UCNPs (30 µg). Following stereotaxic injection, the target site was irradiated by a NIR laser (980 nm, 425 mW/cm2 for 3 cycles of 5 min) to induce the release of Cre from the NPs. After 4 weeks, animals were subjected to a CPP test, where the animal’s preference for one of two regions with contrasting visual and tactile cues in a cage was monitored for 3 days using an automated mouse tracking system. On day 4, whenever the mouse crossed into the non-preferred region of the cage, the VTA was activated with blue light; on the final test day, the animals were re-introduced into the cage to assess changes in preferences. b Quantification of Cre-UCNPs accumulated in the brain of mice was performed in dried tissue by ICP-MS at days 0, 3, and 7 post stereotaxic administration. Results are Mean ± SEM (n = 4–5 animals). c Local of injection of Cre-UCNPs. d Expression of ChR2-YFP (green) in the TH-positive region (red) of the VTA, as evaluated by immunofluorescence. Of note the differences in fluorescence between left and right VTA (n = 6 animals). e CPP test to evaluate behavioral changes in preference (fold change in the time spend on the non-preferred chamber before and after blue light stimulation). Results are Mean ± SEM, obtained from n = 3 (CRE-UCNPs), or 6 (Negative control and CRE-UCNP + NIR) animals. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test: (*), P = 0.0115. f Expression of c-Fos positive nuclei in the VTA, following stimulation with blue light. Results are expressed as Mean ± SEM (2–5 animals per condition; for each animal, c-Fos was quantified in 2–3 regions in VTA slices, 4-13 fluorescence images per slice) and analyzed by unpaired two-tailed t test: (*), P = 0.024. g Representative confocal images of a mouse treated with Cre-UCNPs showing the expression of c-Fos (gray) and ChR2-YFP (green) in the TH positive region (red) of the VTA, from n = 5 animals. Scale bar = 20 μm.

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