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目的构建APOEε2和APOEε4的真核表达载体,转染APOE敲除鼠神经干细胞,分别建立转染APOEε2和ε4的神经干细胞系。方法以前期克隆得到的APOEε3cDNA为基础,通过定点突变技术得到APOEε2和APOEε4cDNA,亚克隆入表达载体pEGFP-N1,构建pEGFP-N1-APOEε2及pEGFP-N1-APOEε4的真核表达载体,经双酶切和测序确定序列及阅读框正确。以脂质体转染法转染APOE敲除鼠神经干细胞,通过G418筛选,建立转染APOEε2和ε4的神经干细胞系。采用RT-PCR、Westernblot及免疫荧光法检测APOE的表达。结果APOE敲除鼠NSCs转染6h后发出绿色荧光,48h达到高峰,荧光显微镜下观察转染率约(40.0±2.3)%,转染pEGFP-N1-APOE的NSCs在筛选至第10天可见细胞克隆形成。经鉴定保持了自我更新、多向分化保持性,其中分化为神经元的比例为(32.15±2.61)%,各组之间无明显差异。结论成功构建了稳定表达源性APOEε2及ε4的神经干细胞克隆,相应等位基因修饰的神经干细胞能高效表达目的基因蛋白并保留其干细胞特性。
Objective To construct APOEε2 and APOEε4 eukaryotic expression vector and transfect APOE knock-out neural stem cells to establish neural stem cell lines transfected with APOEε2 and ε4, respectively. Methods APOEε2 and APOEε4 cDNAs were obtained by site-directed mutagenesis and subcloned into the expression vector pEGFP-N1. The eukaryotic expression vector pEGFP-N1-APOEε2 and pEGFP-N1-APOEε4 were constructed by double enzyme digestion And sequencing to determine the correct sequence and reading frame. The APOE knockout mouse neural stem cells were transfected by lipofectamine. The neural stem cells transfected with APOEε2 and ε4 were established by G418 screening. APOE expression was detected by RT-PCR, Western blot and immunofluorescence. Results After transfection of APOE knock-out NSCs, green fluorescence appeared and reached the peak at 48h after transfection. The transfection efficiency was about (40.0 ± 2.3)% under the fluorescence microscope. The transfected NSCs transfected with pEGFP-N1-APOE showed cells Clone formation. The self-renewal and multi-directional retentivity were identified. The percentage of differentiated neurons was (32.15 ± 2.61)%. There was no significant difference between the groups. Conclusion The neural stem cell clones stably expressing APOEε2 and ε4 were successfully constructed. The corresponding alleles modified neural stem cells can express the target gene protein efficiently and retain its stem cell characteristics.