利用Cre-LoxP技术构建标记GABA能神经元的转基因小鼠

Constructing transgenic mice to label GABAergic neurons on the based of Cre-Loxp technology

目的:通过Cre-LoxP技术构建转基因小鼠,示踪全脑γ-氨基丁酸(GABA)能神经元分布及形态.方法:使用C57BL/6J-Gad2^(em1(IRES-iCre-pA)Smoc)小鼠(Gad2-ires-cre小鼠)与B6/JGpt-H11^(em1Cin(CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato))/Gpt小鼠(B6-G/R小鼠)杂交产生Gad2^(tdTomato)/non-Gad2^(ZsGreen)子代小鼠,使用CatWalk步态分析系统分析子代小鼠完成全程时间、行走速度和协调指数的差异性;使用激光共聚焦显微镜观察小鼠全脑GABA能神经元的分布情况,并用Imaris软件对GABA能神经元进行3D重塑.结果:CatWalk步态分析结果显示,完成全程时间、行走速度和协调指数在Gad2^(tdTomato)/non-Gad2^(ZsGreen)小鼠与野生型小鼠组间无统计学差异.GABA能神经元主要分布在纹状体(ST)、嗅结节(TU)、大脑脚(CP)、黑质网状部(SNr)及小脑皮层(CbC).ST中GABA能神经元呈椭圆形,并有角状凸起;小脑浦肯野细胞(PC)树突伸入分子层,胞体排列整齐,形成浦肯野细胞层(PCL),轴突穿过颗粒细胞层(GCL),投射到小脑核(CBN).结论:Gad2^(tdTomato)/non-Gad2^(ZsGreen)小鼠运动正常,并可示踪全脑GABA能神经元的分布及形态.

Objective:To construct transgenic mice by Cre-LoxP technology to trace the distribution and morphology of GABAergic neurons in the whole brain.Methods:Used C57BL/6J-Gad2^(eml(IRESlCrepA)Smoc) mice(Gad2-ires-cre mice)and B6/JGpt-H11^(emlCin(CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato))/Gpt mice(,B6-G/R mice)hybrid to produce Gad2^(tdTomato)/non-Gad2^(ZsCreen) mice,and CatWalk used to analyze the difference in whole journey time(Run duration,s),average walking speed(Speed,cm/s)and coordination index(Regularity index,%)between wild-type(WT)and Gad2^(tdTomato)/non-Gad2^(ZsGreen) mice.Laser scanning confocal microscope was used to observe the distribution of GABAergic neurons in the whole brain of mice,and Imaris software used to remodel GABAergic neurons in 3D.Results:Cat Walk analysis showed that the time to complete the whole process,walking speed and coordination index was not statistically different between the WT and Gad2^(tdTomato)/non-Gad2^(ZsGreen) mice group.GABAergic neurons were mainly distributed in the striatum(ST),olfactory tubercle(TU),cerebral peduncle(CP),substantia nigra reticulata(SNr)and cerebellar cortex(CbC).GABAergic neurons in ST were elliptical with horn-like protrusions,cerebellar Purkinje cell(PC)dendrites extended into the molecular layer,the cell bodies arranged neatly to form the Purkinje cell layer(PCL),and the axons passed through the granule cell layer(GCL),which projected to the cerebellar nucleus(CBN).Conclusion:Gad2^(tdTomato)/non-Gad2^(ZsGreen)mice exercised normally and could trace the distribution and morphology of GABAergic neurons in the whole brain.