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. 2023 Dec 19;14:8452. doi: 10.1038/s41467-023-44184-0

Fig. 5. In vitro gene editing of AEP-derived alveolar organoids via AAV6.2FF-Cre.

Fig. 5

A AAV6.2FF-Cre experimental set-up. Live/CD31−/CD45−/CD326+(EpCAM+)/TdTomato+(Axin2+) cells (AEPs) sorted from mice with the R26REYFP allele (Axin2creERT2-tdT; R26REYFP) were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. B H&E of 5 µm sections of FFPE day 29 AAV6.2FF-Cre-treated organoids, exhibiting morphology and structural complexity similar to untreated/control organoids (Fig. 1B). C Whole-well brightfield and GFP images of day 29 organoids (untreated vs. AAV6.2FF-Cre-treated) at MOI = 1000. D Comparison of cells treated with an MOI of 1000, 10000, 20000. Quantification of day 32 organoids (n = 3 wells per condition) showing that an MOI of 1000 causes recombination in organoids without major effects on colony-forming efficiency (CFE). E Quantification (n = 5 wells per condition) showing that an MOI = 1000 induces significant levels of recombination without effect on organoid number or size. F Whole-mount immunofluorescence of day 32 AAV6.2FF-Cre-treated AEP-derived organoids (same experimental set-up as Fig. 2). White box highlighting untargeted epithelial cells (YFP−/GFP−) next to a targeted (YFP+/GFP+) organoid in the same well, supporting clonal expansion of AAV6.2FF-Cre-treated cells. Note: EYFP was stained for using anti-GFP antibodies and imaged (whole-well images) using GFP filter cubes. Data throughout the figure represents greater than 5 biological replicates with 3 technical replicates per experiment. [Scale bars = 50 µm]. AAV Adeno-associated virus, MOI multiplicity of infection. Box and whiskers plot shows box from 25–75% with central line at median and whiskers to the maximum and minimum points; all data points are graphed. Source data are provided within the Source Data file. Schematics created with Biorender.com.