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• W4366995850 abstract "To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.利用RPS6启动子实现凝血因子的稳定表达.筛选得到更优启动子，为血友病基础研究和基因治疗提供更有力的工具。.用生物信息学方法分析高丰度表达持家基因的启动子，遴选潜在候选启动子；构建 GFP报告基因载体，以EF1α启动子为对照，考察新型启动子载体的包装效率及报告基因的转录和活性；装载F9 基因，考察候选启动子的活力。.筛选得到最具潜力的RPS6启动子；RPS6pro-LV和EF1αpro-LV在慢病毒包装上没有差异，二者病毒滴度一致。在293T细胞中，RPS6pro-LV和EF1αpro-LV的转导效率、平均荧光强度与慢病毒剂量成正比。两种启动子在不同类型细胞中的转导效率均呈现293T>HEL>MSC的规律；相较于EF1αpro-LV，RPS6pro-LV在MSC细胞中具有更高的荧光强度；对感染两种启动子病毒的HEL细胞进行长期培养，发现RPS6pro-LV具有更稳定的活性。K562细胞RT-qPCR、Western blot及细胞培养上清FIX活性（FIX∶C）检测结果显示，EF1α-F9、RPS6-F9组FIX表达均高于空载对照组，EF1α-F9与RPS6-F9组之间FIX表达量无显著差异。.经筛选和优化，得到一个可广泛用于外源基因表达的新型启动子，通过长期培养和活性基因表达证实该启动子的高度稳定性和高效活力，为血友病基础研究和临床基因治疗提供了一个有力工具。." @default.
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• W4366995850 date "2023-04-01" @default.
• W4366995850 modified "2023-09-27" @default.
• W4366995850 title "[Stable Expression of Coagulation Factors by RPS6 Promoter]." @default.
• W4366995850 doi "https://doi.org/10.19746/j.cnki.issn.1009-2137.2023.02.026" @default.
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