Aladdin A, Sahly N, Faty R, Youssef M M, Salem T Z
Biomedical Sciences Program, University of Science and Technology, Zewail City of Science and Technology, October Gardens, 6th of October City, Giza 12578, Egypt.
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt.
Gene. 2021 May 20;781:145541. doi: 10.1016/j.gene.2021.145541. Epub 2021 Mar 2.
Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.
理解启动子在非宿主细胞中的作用机制是复杂的。尽管如此，在进行基因表达调控研究时，了解这一过程至关重要。本研究始于构建一种穿梭载体，该载体将巨细胞病毒（CMV）启动子和昆虫痘病毒IE2（OpIE2）启动子串联排列，以便分别在哺乳动物细胞和昆虫细胞中实现基因表达。在这个系统中，发现OpIE2昆虫病毒启动子5'端的抑制区域会阻断CMV启动子在哺乳动物细胞中的活性。最初，将OpIE2启动子克隆到CMV启动子的下游和增强绿色荧光蛋白（EGFP）报告基因的上游。将构建好的穿梭载体导入昆虫细胞和哺乳动物细胞后，观察到CMV启动子在哺乳动物细胞中的活性显著下降。为了增强CMV启动子的活性，对穿梭载体进行了多项修饰，包括定点诱变以去除下游启动子（OpIE2）中的所有甲硫氨酸密码子，分离两个启动子以消除它们之间转录干扰的影响，最后，在OpIE2启动子序列中确定了一些抑制区域。去除这些抑制区域后，昆虫细胞和哺乳动物细胞均维持了高表达水平。总之，构建了一种在没有杆状病毒感染或基因表达的情况下能在哺乳动物细胞系和昆虫细胞系中高效工作的穿梭载体。此外，该穿梭载体可作为一个平台，进一步研究这种抑制作用的原因，这可能为昆虫和哺乳动物宿主中转录和启动子的作用模式提供新的见解。
