ObjectiveTo construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. MethodsMb1-cre transgenic mice were crossed with Spi1^flox/flox mice. PCR combined with agarose gel electrophoresis was performed for genotyping， and Mb1-cre×Spi1^flox/flox homozygous knockout mice were screened out. Flow cytometry was used to detect the expression of PU.1 protein in various immune cell subsets to verify the specificity of gene knockout. ResultsPCR identification results showed that when using flox primers for detection， only a 220 bp band was amplified， indicating a genotype of Spi1^flox/flox in the mice. When using Mb1-Cre primers for detection， a 383 bp band was amplified， indicating a genotype of Mb1-cre×Spi1^flox/flox in the mice； Flow cytometry results showed that compared to Spi1^flox/flox mice， Mb1-cre×Spi1^flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells， peripheral blood mononuclear cells， and spleen-derived B cells. Moreover， PU.1 exhibited normal expression in other immune cells， such as CD4⁺ T cells and macrophages. ConclusionBy utilizing the Cre/LoxP system and CRISPR/Cas9 technology， this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene， providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.