中国癌症防治杂志2023，Vol.15Issue(6)：630-636,7.DOI:10.3969/j.issn.1674-5671.2023.06.06
巨噬细胞条件性GP73基因敲除小鼠的构建及鉴定
Construction and identification of macrophage-conditional GP73 gene knockout mice
摘要
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 GP73flox/+ mice were constructed based on the Cre/LoxP recombination system.GP73flox/flox mice were obtained by self-cross of GP73flox/+mice.GP73flox/flox mice were crossed with Lyz2-Cre+mice to obtain GP73flox/+Lyz2-Cre+ mice,and then crossed with GP73flox/flox mice to obtain the macrophage-conditional GP73 gene knockout mice,referred to as GP73flox/floxLyz2-Cre+mice(MKO mice).The GP73flox/floxLyz2-Cre-(GP73fl/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 GP73fl/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 GP73fl/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 GP73fl/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 GP73gene 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.关键词
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Key words
Golgi protein 73/Macrophage/Cre/LoxP recombination system/Conditional knockout/Genotype identification/Mouse model分类
医药卫生引用本文复制引用
黄庆,甄兰,赵桂霖,赵汝舟,叶新萍,吴飞翔..巨噬细胞条件性GP73基因敲除小鼠的构建及鉴定[J].中国癌症防治杂志,2023,15(6):630-636,7.基金项目
广西重点研发计划项目(桂科AB16380242) （桂科AB16380242）
区域性高发肿瘤早期防治研究教育部重点实验室(广西医科大学)自主课题(GKE-ZZ202216) （广西医科大学）
