Abstract: Objective To establish oviductal epithelial organoids derived from Cas9⁃EGFP mice and perform gene editing. Methods Oviductal epithelial cells were isolated from Cas9⁃EGFP mice via enzymatic digestion and cultured in organoid⁃specific medium. The proliferation rates and size progression of organoids were monitored via continuous imaging. Cas9⁃EGFP expression was observed using fluorescence microscopy. Immunofluorescence staining was performed to confirm the origin of reproductive system and assess the expression of ciliated cell markers. Lentivirus carrying sgTrp53 was used to infect Cas9⁃EGFP mice oviductal epithelial organoids, with editing efficiency was validated by T7E1 assay. Results The abdominal cavity of mice was accessed through a midline incision, revealing coiled oviducts located between the uterine horns and ovaries. Oviductal epithelial organoids derived from Cas9⁃EGFP mice assembled into cystic structures by day 2 of culture, reaching diameters of 400 μm by day 7, with stable Cas9 protein expression. Immunofluorescence confirmed their origin from the female murine reproductive system, and expression of ciliated cell markers. Following lentiviral transduction with sgTrp53, Trp53 mRNA expression was significantly reduced in edited organoids compared to unedited controls (P=0.021). Conclusions Oviductal epithelial organoids are successfully established from Cas9⁃EGFP mice, and CRISPR⁃Cas9⁃mediated gene editing was achieved in this model system.

Key words: Oviducts, Organoids, Gene editing