关键词: line-height:18px, 高尔基体蛋白73；巨噬细胞；Cre/LoxP重组系统；条件性敲除；基因型鉴定')">">高尔基体蛋白73；巨噬细胞；Cre/LoxP重组系统；条件性敲除；基因型鉴定, 小鼠模型

Abstract: Objective To construct and identify macrophage⁃conditional Golgi protein 73(GP73) gene knockout mice, and to provide an animal model for exploring the effect of GP73 on the occurrence and development of neoplastic diseases by regulating the function of macrophages. Methods The GP73 ^flox/+ mice were constructed based on the Cre/LoxP recombination system. GP73^flox/flox mice were obtained by self⁃cross of GP73^flox/+mice. GP73^flox/flox mice were crossed with Lyz2⁃Cre+ mice to obtain GP73^flox/+Lyz2⁃Cre+ mice, and then crossed with GP73^flox/flox mice to obtain the macrophage⁃conditional GP73 gene knockout mice, referred to as GP73^flox/floxLyz2⁃Cre+ mice(MKO mice). The GP73^flox/floxLyz2⁃Cre- (GP73^fl/fl) mice were used as the control mice. The genotypes of flox and Cre were identified by PCR and agarose gel electrophoresis. Real⁃time fluorescence quantitative PCR (qPCR) and Western blot were used to verify the knockout efficiency and tissue specificity of macrophages GP73 at mRNA and protein levels, respectively. The ratio of tissue weight to body weight was calculated to analyze the growth and development of mice, and the blood biochemical indicators of mice were detected. Results The successful construction of macrophage⁃conditional GP73 gene knockout mice was confirmed by genotype identification, mRNA, and protein level analysis. qPCR tests showed that, compared with GP73^fl/fl mice, the GP73 mRNA levels in bone marrow⁃derived macrophages (BMDMs) and peritoneal macrophages (PM) of MKO mice were decreased (P<0.0001). Western blot detection showed that compared with GP73^fl/fl mice, the expression levels of GP73 protein in BMDMs and PM in MKO mice were significantly decreased, though no significant differences were found in the expression levels of GP73 protein in liver, kidney and thymus tissues. Compared with GP73^fl/fl mice, the ratio of body weight to tissue weight of heart, liver, spleen, lung, kidney, brown adipose and white adipose in MKO mice was not significantly different, and there was no significant morphological difference among the tissues. The results of blood biochemical indicators showed that there were no significant difference in blood biochemical indexes between the MKO mice and GP73fl/fl mice (P>0.05). Conclusions  The mouse model of macrophage⁃conditional GP73 gene knockout is successfully constructed, providing a valuable animal model for the further study of the role and mechanism of GP73 in regulating macrophage in neoplastic diseases.oxP recombination system; Conditional knockout; Genotype identification; Mouse model

Key words: Golgi protein 73')">Golgi protein 73, Macrophage, Cre/LoxP recombination system, Conditional knockout, Genotype identification, Mouse model