Introduction: The multiple protein expression using a single vector cassette is a very useful technique for generation of geneticallymodified pigs for xenotransplantation. The internal ribosomal entry site (IRES) element has a pitfall that the second gene at downstream is not expressed well relative to the first gene in mammalian cells. Although the second gene is well expressed in mammalian cells by the viral 2A vector system, the different destination of target proteins may affect the mutual expression level. Here, we investigated whether the upstream and downstream gene of the viral 2A segment is well expressed regardless of their final destination. Materials and methods: We cloned various cassettes including two genes into mammalian expression vector, pcDNA3.1 and transfected each of them into pig embryonic fibroblasts by electroporation. The protein expression levels were checked by western blotting or flow cytometry using extra- or intra-staining protocol. Results: In cassette # 1 and 2, the protein of EGFP and HA-HO1 at upstream of IRES element was expressed well. But, the protein expression of HA-HO1 and EGFP at downstream of IRES element was very low or not detected. In cassette # 3, 4, 5 and 6 the protein of EGFP, HA-HO1, EPCR and TM was expressed well regardless of their location in the vector. In cassette # 7 and 8, the protein of TM was expressed well regardless of the location. But, the protein expression of EGFP at the downstream of T2A segment was lower than the upstream EGFP. In cassette # 9, the protein expression level of the upstream gene, CD46, was extremely low and the the downstream gene, TM, was not expressed. In cassette # 10, the both of genes were expressed well. Overall, these data demonstrated that the multicistronic expression system using the viral 2A segment works better than the system using IRES element in mammalian cells, and that the protein expression level of target genes in the viral 2A system, was almost same regardless of their location in the vector even if they go toward different destination in cells. However, the protein expression level of the first gene determines the expression level of the second gene.Table: [Vector cassettes using in this study]Conclusion: The multicistronic expression system using the viral 2A segment is a useful system for making good expression of multiple genes, if protein expression level of the first gene is adequate. Therefore, a 2A vector system is promising for making genetically-modified pigs for multiple target genes for xenotransplantation. (Abbreviations: HA-HO1: Heme oxygenase 1 tagged with HA; TM: Thrombomodulin; EPCR: Endothelial protein C receptor; T2A: 2A segment from Thosea asigna virus; CP: Cytosolic protein; MP: Membrane protein)