Lentiviral infection is often used to integrate genetic material into cells to stably express transgenes of interest. Depending on the location of integration into the host genome, readthrough expression of the lentiviral cargo can occur via an upstream endogenous promoter, which is typically an unwanted phenomenon because it can result in dysfunctional expression. The purpose of this study was to demonstrate that readthrough expression can be a wanted phenomenon for expressing functional proteins while at the same time reducing the size of the lentiviral transfer plasmid. Readthrough expression was used to generate HEK293 cell lines stably expressing fluorescent reporter proteins, reporter protein-antibiotic resistance fusion proteins for selection, and the vascular endothelial growth factor receptor 2. The generated proteins were all functional, as demonstrated by their ability to fluoresce, confer antibiotic resistance, and participate in receptor-mediated signalling, respectively. Therefore, we suggest that the mechanism of readthrough expression may have further applications in the expression of larger genes or genetic circuits (e.g. cell-based therapeutics), where the lentiviral cargo limit is stretched to the maximum.

慢病毒感染通常用于将遗传物质整合到细胞中以稳定表达感兴趣的转基因。根据整合到宿主基因组的位置，慢病毒货物的通读表达可以通过上游内源启动子发生，这通常是一种不希望的现象，因为它可能导致表达功能障碍。本研究的目的是证明通读表达可能是表达功能蛋白的一种理想现象，同时减少了慢病毒转移质粒的大小。通读表达用于生成稳定表达荧光报告蛋白的 HEK293 细胞系，用于选择的报告蛋白-抗生素抗性融合蛋白和血管内皮生长因子受体 2。生成的蛋白质都是功能性的，正如它们分别发出荧光、赋予抗生素抗性和参与受体介导的信号传导的能力所证明的那样。因此，我们认为通读表达的机制可能在更大的基因或遗传回路（例如基于细胞的治疗）的表达中具有进一步的应用，其中慢病毒货物限制被拉伸到最大值。