CX3 CR1^(GFP)报告基因小鼠的构建、鉴定及效率检测

Construction,identification and efficiency detectionof CX3CR1 GFP reporter gene mice

目的 基于Cre/Loxp系统构建CX3CR1^(GFP)基因小鼠,并分析CX3CR1^(GFP)表达效率。方法 通过以限制酶为基础的克隆技术设计靶向载体,用线性化的靶向载体转染胚胎干细胞(embryonic stem,ES),将筛出缺失新霉素抗性基因(neomycin resistance gene,neo)的重组ES细胞克隆注入囊胚中产生嵌合CX3CR1^(+/GFP)小鼠,与野生型小鼠(wild type mice,WT)交配,反复回交得到CX3CR1^(GFP/GFP)小鼠。分别提取WT和CX3CR1^(GFP)小鼠DNA及mRNA,使用琼脂糖凝胶电泳鉴定基因型,RT-qPCR检测小鼠不同组织中CX3CR1的表达水平;Western blot分析GFP蛋白对外周血中的单个核细胞(peripheral blood mononuclear cell,PBMC)及不同组织中CX3CR1的表达;流式细胞术检测骨髓中免疫细胞的标记效率;免疫荧光观察小鼠不同组织中GFP的表达情况。结果琼脂糖凝胶电泳结果表明,转基因小鼠基因型为CX3CR1^(GFP/GFP)纯合子。RT-qPCR和Western blot结果表明,增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)已靶向取代CX3CR1基因,因此在CX3CR1^(GFP)小鼠中CX3CR1表达极低,而GFP表达明显上调;流式细胞术和免疫荧光显示,GFP有效标记CX3CR1^(GFP)小鼠,在不同组织及不同细胞均有表达且表达具有差异。结论 本研究构建并鉴定CX3CR1^(GFP)基因报告小鼠,且GFP在小鼠体内稳定表达。为进一步揭示CX3CR1在免疫调节中的潜在机制研究提供动物模型基础。

Aim To construct CX3CR1^(GFP) transgenic mice based on the Cre/Loxp system,and to analyze the expression efficiency of CX3CR1 GFP.Methods Targeted vectors were designed using restriction enzyme-based cloning technology to create a linearized targeted vector for transfecting embryonic stem cells(ES).The ES cells with a deletion of the neomycin resistance gene(neo)were then cloned into blastocysts to generate chimeric CX3CR1^(+/GFP) mice.These mice were subsequently bred with wild-type mice(WT),and repeated backcrossing was performed to obtain CX3CR1 ^(GFP/GFP) mice.DNA and mRNA from WT and CX3CR1^(GFP) mice were extracted and genotyped using agarose gel electrophoresis.The expression level of CX3CR1 in various tissues of the mice was detected by RT-qPCR.Western blot analysis was used to analyze the expression of GFP protein in peripheral blood mononuclear cells(PBMC)and various tissues.The labeling efficiency of immune cells in bone marrow was detected by flow cytometry.The expression of GFP in different mouse tissues was observed by immunofluorescence.Results The results of agarose gel electrophoresis showed that the transgenic mouse genotype was CX3CR1 ^(GFP/GFP) homozygote.RT-qPCR and Western blot showed that EGFP were targeted to replace CX3CR1 gene,so CX3CR1 expression was very low in CX3CR1^(GFP) mice,while GFP expression was significantly upregulated.Flow cytometry and immunofluorescence showed that GFP effectively marked CX3CR1^(GFP) mice,expressed in various tissues and cells with different expression levels.Conclusion This study constructs and identifies the CX3CR1^(GFP) genetic reporter mice,and GFP is stably expressed in mice,which provides a foundation for further research into the potential mechanisms of CX3CR1 in immune regulation.