Construction of a Cc10 Gene Knockout Mouse Model and Phenotypic Analysis of Airway Inflammation
LONG Jie1, WEI Dandan1, WU Quanlong1, ZHAO Ziying1, ZHANG Yaya1, CHEN Yanjiao1,2, YANG Yongqing1,2, XU Yudong1,2*
This study aimed to construct a Cc10 gene knockout mouse model and analyzed the airway inflammatory phenotype of Cc10-deficient mice using a HDM (house dust mite)-induced asthma model. CRISPR/Cas9 technology was employed to design and transcribe sgRNA (single guide RNA) targeting exon 2 of the Cc10 gene, along with Cas9 expression vectors in vitro. Fertilized eggs were microinjected with these components, resulting in the generation of F0 knockout mice. The allergic asthma model was then developed in these mice by sensitization and challenge with HDM. The impact of Cc10 gene deficiency on airway inflammatory phenotypes was evaluated by assessing leukocyte counts and their classified counts, type 2 inflammation-associated cytokines in BALF (bronchoalveolar lavage fluid), performing PAS staining on lung tissues, and measuring Muc5ac mRNA expression levels. PCR amplification and sequencing confirmed the successful knockout of the Cc10 gene. Western blot analysis demonstrated the absence of CC10 protein expression in the lung tissues of Cc10–/– mice. The total leukocyte count in BALF of Cc10–/– asthmatic mice was significantly higher than that of the WT (wild-type) counterparts, with notable increases observed in neutrophils, lymphocytes, eosinophils, macrophages, and basophils. The levels of type 2 inflammation-associated cytokines IL-4, IL-5, and IL-13 were significantly higher in the BALF of Cc10–/– asthma model mice than in the WT asthma model group.PAS staining of lung tissues revealed that the Cc10–/– asthmatic mice exhibited significant pathological changes, including thickened bronchial walls and goblet cell hyperplasia, with PAS staining scores markedly higher than those in WT asthmatic mice. PCR analysis showed that Muc5ac mRNA expression levels in the lung tissues of Cc10–/– asthmatic mice were significantly elevated compared to WT counterparts. Collectively, this study successfully constructed a Cc10 gene knockout mouse model and demonstrated that Cc10 deficiency exacerbates airway inflammation and enhances mucus secretion in asthma. These findings suggest that CC10 plays a crucial role in regulating airway inflammation, and the model provides a valuable tool for investigating the mechanisms of CC10 in asthma and other respiratory diseases.
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