Abstract: Objective To establish a mouse duodenal mucosal epithelial organoid model with conditional knockout of dopamine D5 receptor (D₅R) in intestinal mucosal epithelium. Methods Villin-Cre^ERT2+/+D₅R-flox^+/+ C57BL/6J mice were utilized to isolate duodenal crypts for organoid culture. D₅R conditional knockout (CKO) was induced via the Cre-loxP system by 4-hydroxytamoxifen administration. The structural characteristics of duodenal mucosal epithelial organoids and the knockout efficiency of D₅R were evaluated through morphological observation, immunofluorescence staining, quantitative PCR (qPCR), and Western blot assays. Results A duodenal mucosal epithelial organoid model with conditional D₅R knockout was successfully established. Morphological examination revealed that the organoids exhibited structural features similar to the in vivo duodenal mucosal epithelium. Immunofluorescence staining confirmed a significant reduction in D₅R expression following 4-hydroxytamoxifen induction,with fluorescence intensity decreasing from 0.904±0.061 to 0.385±0.020 (n=6, P＜0.001). qPCR analysis demonstrated a marked decrease in D₅R mRNA levels from 2.483±0.204 to 0.241±0.048, and Western blot showed a significant reduction in protein expression from 0.923±0.075 to 0.569±0.032 (n=6, P＜0.001). Conclusions A conditional D5R knockout organoid model derived from duodenal mucosal epithelium is successfully developed, which faithfully recapitulates the physiological characteristics of duodenal mucosa. This model provides a reliable in vitro platform for elucidating the regulatory mechanisms of D5R in duodenal epithelial functions.

Key words: organoid, duodenum, intestinal mucosal epithelium, dopamine D5 receptor