红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致

Integration and expression of GFP gene directed by the erythroid-specific element in transgenic mice

To study the relationship between integration status and expression level of the foreign gene, copy number of the foreign green fluorescence protein (GFP) gene directed by the erythroid-specific elements (HS2 and HS3) in transgenic mice were determined by real-time quantitative PCR, and the GFP expression level in peripheral blood was measured by fluorescence-activated cell sorter (FACS) analysis and was observed under a fluorescence microscope. The integrated sites of foreign gene in two transgenic mice were detected using fluorescene in situ hybridization (FISH). The results showed that the copy numbers of the foreign gene were different and varied greatly in various transgenic mouse lines. The copy number in one transgenic mouse (GFP-19) was as high as 63, and only two copies of foreign gene were detected in another transgenic mouse (GFP-75). Moreover, statistic analysis indicated that the copy numbers was not correlated with the expression level of GFP gene (r=-0.29684,P=0.4379). FISH analysis suggested that the foreign genes were integrated in the different chromosomes of the two transgenic mice, and the intensity of hybridization signals was coincident with the copies of GFP gene. We concluded that the expression level of foreign genes in transgenic mice may rely more on integration sites on chromosomes than copy numbers.