Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
PLoS One. 2013 Jul 29;8(7):e70486. doi: 10.1371/journal.pone.0070486. Print 2013.
In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient.
在猪到灵长类动物的异种移植中，需要多个转基因猪来克服一系列移植排斥反应。通过繁殖或引入几个单顺反子载体来产生多个转基因猪，受到靶基因表达模式的差异的阻碍。为了实现多个基因的同时表达，可以考虑使用来自 Thosea asigna 病毒（T2A）的 2A 肽的多顺反子表达系统作为替代选择。在将 T2A 表达系统应用于猪的产生之前，应该精确评估该系统中多个基因的表达模式。在这项研究中，我们构建了几个双顺反子 T2A 表达载体，它们结合了异种移植领域中常用的靶基因，并将其引入猪成纤维细胞。靶向同一或不同亚细胞区室的蛋白质被有效地表达，而不会影响另一种蛋白质的定位或表达水平。然而，当将低表达效率的基因插入 T2A 序列的上游区域时，与不插入时的表达效率相比，下游基因的表达水平显著降低。该系统中针对一个基因的小干扰 RNA 导致靶基因和另一个基因的显著下调，表明组合到 T2A 表达载体中的多个基因可以被视为单个基因在转录和翻译方面。总之，如果上游基因的表达是有效的，则可以实现下游基因的有效表达。
