基于Cre-LoxP系统的B细胞特异性荧光报告基因小鼠模型的构建及鉴定

Construction and characterization of B cell-specific fluorescent reporter mouse model based on Cre-LoxP system

目的应用Cre^(-)LoxP重组酶系统构建B细胞特异性表达tdTomato蛋白的荧光报告基因小鼠。方法在Gt(ROSA)26Sor(Rosa26)位点插入1个由CAG启动子驱动的报告基团,其LoxP侧翼设计有STOP盒,可阻止tdTomato荧光蛋白表达,经与CD19-Cre工具小鼠交配繁殖获得tdTomato^(CD19-Cre)小鼠,Cre重组酶特异性识别LoxP序列,删除STOP盒,从而使B细胞特异性表达tdTomato荧光蛋白。利用PCR进行基因型鉴定。通过流式细胞术和脑膜免疫荧光分别检测野生型对照组小鼠和tdTomato^(CD19-Cre)荧光报告基因小鼠脾和脑膜B细胞中tdTomato红色荧光蛋白表达。结果基因型为tdTomato^(F/-)CD19-Cre^(+)或tdTomatoF/FCD19-Cre^(+)的小鼠为B细胞特异性表达tdTomato天然红色荧光蛋白的荧光报告基因小鼠。而对照组小鼠基因型为tdTomato^(F/-)CD19-Cre^(-)或tdTomatoF/FCD19-Cre^(-)。流式细胞术测定结果表明,与野生型对照组小鼠相比,tdTomato^(CD19-Cre)荧光报告基因小鼠脾中表达tdTomato荧光蛋白的B细胞百分比均显著增加(P<0.01);在非B细胞群中仅检测到少量的tdTomato+细胞。且在滤泡型B细胞、边缘区B细胞、T1 B细胞、T2 B细胞和成熟B细胞等B细胞亚群细胞表面tdTomato荧光蛋白均能稳定表达(P<0.01),百分比分别为(88±8)%,(80±9)%,(86±9)%,(92±6)%和(89±7)%。通过与CD45共定位,在tdTomato^(CD19-Cre)荧光报告基因小鼠脑膜中也检测到tdTomato红色荧光蛋白表达。结论成功构建B细胞特异性表达tdTomato荧光蛋白的荧光报告基因小鼠模型,且tdTomato荧光蛋白可在外周免疫器官和中枢神经系统稳定表达。

OBJECTIVE To construct a mouse model with B cell-specific tdTomato fluorescent reporter using the Cre^(-)LoxP(Cre recombinase-locus of x-over,P1)technique.METHODS A reporter group driven by the CAG promoter was inserted into the Gt(ROSA)26Sor(Rosa26)site,and a LoxPflanked STOP cassette was designed to block the expression of the tdTomato red fluorescent protein.Robust tdTomato fluorescent protein expression depended on the presence of Cre recombinase in cells.The B cell-specific tdTomato fluorescent model mice were obtained by mating with CD19-Cre tool mice.Cre recombinase specifically recognized the LoxP sequence and deleted the STOP cassette so that B cells specifically expressed tdTomato fluorescent protein.Genotyping of mice was determined by PCR analysis with mouse-tail DNA.The expression of tdTomato red fluorescent protein in B cells of the spleen and dura of littermate controls and tdTomato^(CD19-Cre)mice was detected by flow cytometry and immunofluorescence.RESULTS The mice with the genotype tdTomato^(F/-)CD19-Cre^(+)or tdTomatoF/FCD19-Cre^(+)were fluorescent reporter mice that specifically expressed native tdTomato red fluorescent protein.The genotype of the control group was tdTomato^(F/-)CD19-Cre^(-)or tdTomatoF/FCD19-Cre^(-).Compared with littermate wild type controls,the expression of B cell-specific tdTomato red fluorescence increased sig⁃nificantly in tdTomato^(CD19-Cre)fluorescent mice(P<0.01).Only a very small number of tdTomato+cells were detected in the non-B cell population,and the fluorescent protein of tdTomato was stably expressed in follicular B,marginal zone B,T1 B,T2 B and mature B cell subsets(P<0.01).The percentages were(88±8)%,(80±9)%,(86±9)%,(92±6)%and(89±7)%,respectively.The tdTomato fluorescent protein was also detected in the meninges of tdTomato^(CD19-Cre)mice via co-localization with CD45.CONCLU⁃SION A mouse model with B cell-specific tdTomato fluorescent protein is constructed,and the tdTomato fluorescent protein is stably expressed in the peripheral immune organs and central nervous system.