摘要
目的探讨C-C趋化因子受体2型(CCR2)基因敲除鼠的繁育和基因型分析,验证聚合酶链式反应(PCR)法对CCR2基因敲除鼠基因型检测的适用性。方法将引进的CCR2纯合雄性小鼠和野生型雌性小鼠进行交配繁殖出子一代,获得的F1代杂合子小鼠继续进行交配,待小鼠2周龄时剪尾部组织提取DNA,PCR法扩增目的基因片段,琼脂糖凝胶电泳进行基因型结果判定。通过遗传杂交,提高产生携带CCR2基因敲除纯合子后代比例,采用Western blot技术针对主要免疫细胞及关键脏器验证后代小鼠中CCR2基因的敲除效果,使用流式细胞术针对主要免疫细胞检测CCR2基因的敲除对免疫系统功能是否有影响。结果成功繁育并鉴定了CCR2基因敲除鼠,得到了3种基因型的F2代小鼠:CCR2^(+/+)、CCR2^(+/-)、CCR2^(-/-)。通过PCR法鉴别了子代基因型,Western blot显示CCR2基因敲除小鼠中CCR2蛋白表达极低。流式分析表明,CCR2基因敲除会减少小鼠脾脏来源T细胞中CD4^(+)T和Th1型细胞表达,但不影响巨噬细胞功能。结论正确的繁殖和鉴定是得到纯合CCR2基因敲除鼠的重要途径,PCR法鉴别小鼠基因型具有简便、快捷、可靠的特点。
Objective To explore the breeding and genotyping of CCR2 knockout mice,and to verify the applicability of the polymerase chain reaction(PCR)method for genotype detection of CCR2 knockout mice.Methods The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation,the obtained F1 generation heterozygous mice were continued to be mated.DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks,the target gene fragment was amplified by PCR,and the genotypic results were determined by agarose gel electrophoresis.The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses,the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs,and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells.Results CCR2 knockout mice were successfully bred and characterized,and three genotypes of F2 generation mice were obtained:CCR2^(+/+),CCR2^(+/-),and CCR2^(-/-).The offspring genotypes were identified by PCR,and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice.Flow analysis showed that CCR2 knockdown reduced the expression of CD4^(+)T and Th1 cells in mouse spleen-derived T cells,but did not affect macrophage function.Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice,and PCR method for identifying mouse genotypes is simple,fast and reliable.
作者
张慧茹
王安琪
刘崇
周园园
薛慧
涂佳杰
Zhang Huiru;Wang Anqi;Liu Chong;Zhou Yuanyuan;Xue Hui;Tu Jiajie(Institute of Clinical Pharmacology,School of Pharmaceutical Sciences,Anhui Medical University,Hefei 230032)
出处
《安徽医科大学学报》
北大核心
2025年第7期1167-1172,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:82373877)。
