AIM: This study utilized CRISPR/Cas9 technology to create Retnlb floxp knock-in mice, followed by the application of the Cre-LoxP recombination system to generate intestinal epithelial-specific Retnlb gene knockout mice (Retnlb-CKO). This model was developed to investigate the pathogenic mechanisms of Retnlb in inflammatory bowel disease. METHODS: Female and male C57BL/6N mice, aged 8 weeks with the Retnlbflox/+ genotype, were housed together for breeding. Offsprings were screened to identify those with the Retnlbflox/flox genotype. These mice were then crossed with Vil1-Cre transgenic mice, which express Cre recombinase specifically in intestinal epithelial cells, resulting in Retnlbflox/+, Cre+ mice. Subsequent crosses between Retnlbflox/+, Cre+ mice and Retnlbflox/flox mice produced Retnlbflox/flox, Cre+ mice (Retnlb-CKO). Six 8-week-old Retnlbflox/flox, Cre+ mice and their littermate Retnlbflox/flox mice were selected for experiments. RT-qPCR and immunohistochemistry were used to assess Retnlb mRNA and protein levels in colonic epithelium. Phenotypic observations included body length, weight, diet, and reproductive capability. Tissue-to-body weight ratios were calculated to analyze growth and development. Intestinal barrier integrity and colonic expression of inflammatory factors were evaluated. RESULTS: The conditional gene knockout mouse model with specific deletion of Retnlb in intestinal epithelial cells was successfully established and validated through genetic identification, mRNA and protein analysis. Compared to Retnlbflox/flox mice, Retnlb-CKO mice exhibited no significant differences in body length, weight, diet, or reproductive capability. There were no differences in the ratios of heart, liver, spleen, lung, kidney, and colon weight to body weight, nor were there morphological differences in various tissues. However, the mRNA expression of tight junction proteins ZO-1, Occludin, and Claudin3 in colon tissues of Retnlb-CKO mice was significantly reduced( P<0. 01). PAS staining and immunohistochemistry revealed a significant decrease in the number of goblet cells and lysozyme-positive cells in the colon tissues of Retnlb-CKO mice (P<0. 01). HE staining showed no obvious pathological change in colon tissues of Retnlb-CKO mice. RT-qPCR further demonstrated a significant downregulation of pro-inflammatory factors NLRP3, interleukin-6(IL-6), IL-1β, and tumor necrosis factor-α(TNF-α) in colon tissues (P<0. 01), along with significant downregulation of inflammation signaling pathway proteins TLR4, MyD88, and NF-κB (P<0. 01). CONCLUSION: A conditional colon epithelial cell Retnlb gene knockout mouse model was successfully constructed and validated. The absence of Retnlb in colon cells led to impaired intestinal barrier function, decreased mRNA expression of pro-inflammatory factors in colon tissue, and downregulation of mRNA expression of inflammatory pathway proteins TLR4, MyD88, and NF-κB. [ABSTRACT FROM AUTHOR]