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.
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