〔Abstract〕 Objective To construct myeloid-specificSpi1gene knockout mice and analyze their genotypes, so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets. Methods According to the principle of CRISPR/Cas9 technology and Cre/LoxP system, sgRNA and Donor vectors were designed and constructed. The transcript of Exon 2(Exon 2) was used as the knockout region, andLoxpelements were placed on both sides of Exon 2. Cas9 protein, sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice, the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice, and F0 generation was obtained after 19～20 days. Positive F0 mice were mated with C57BL/6J mice to obtain stable F1Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtainSpi1flox/floxmice.Spi1flox/floxmated withLyz2-Cre+mice to obtainSpi1flox/+/Lyz2-Cre+mice, and then mated withSpi1flox/flox, theSpi1flox/flox/Lyz2-Cre+mice were myeloid-specificSpi1gene knockout(KO) mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice. DNA of WT and KO mice was extracted, and the genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene, Spi-1/purine rich box-1(PU.1) in immune cells of WT and KO mice. Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer wasSpi1flox/floxhomozygote, and the genotype of mice with 700 bp amplified byLyz2-Cre primer wasLyz2-Cre+. Western blot showed that compared with WT group, the protein PU.1 was not expressed in bone marrow-derived macrophages(BMDMs) and peritoneal macrophages(PM) in KO group(P<0.01). There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice. The results of PCR and Western blot showed that myeloid-specificSpi1KO mice were successfully constructed. Conclusion The myeloid-specificSpi1gene KO mice are successfully constructed and identified, which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.