Abstract: 【Abstract】 Objective To construct a serine protease inhibitor Kazal type-5 （Spink5） conditional knockout mouse model, and to identify its phenotype. Methods B cell-specific Spink5 conditional knockout mice of genotype Mb1cre/+Spink5floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated protein 9 （Cas9） technology, and served as the knockout group. Mice of genotype Mb1+/+Spink5floxp/floxp served as the control group. The mice of genotype Mb1cre/+Spink5floxp/floxp or Mb1+/+Spink5floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor （LEKTI） was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group （0.66 ± 0.11, t = 9.99, P < 0.001）, and that in the B lymphocytes in the control group （1.08 ± 0.13, t = 13.78, P < 0.001）. Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group （29.71 ± 3.63 μm, t = 5.05, P = 0.002） and that of normal skin tissues in the control group （12.42 ± 2.21 μm, t = 6.74, P < 0.001）. Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues （46.21 ± 1.21）, non-lesional skin tissues （46.62 ± 2.13） in the knockout group and normal skin tissues in the control group （47.69 ± 1.71, P > 0.05）. Conclusion The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Key words: Netherton syndrome, Mice, knockout, B lymphocytes, Spink5 gene, LEKTI, Skin barrier dysfunction