Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT
- 150 Downloads
Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.
KeywordsXenotransplantation T2A-based bicistronic vector GGTA1-knockout Human complement regulatory proteins
This work was supported by grants from Major Program on Basic Research Projects of Yunnan Province (Grant No. 2014FC006), the National Genetically Modified Organisms Breeding Major Projects (Grant No. 2016ZX08009-003-006) and the National Natural Science Foundation of China (Grant No. 31560637).
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no competing interests.
- 12.Dor, F. J., Kuwaki, K., Tseng, Y. L., Shimizu, A., Houser, S. L., Yamada, K., et al. (2005). Potential of aspirin to inhibit thrombotic microangiopathy in alpha 1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons. Transplantation Proceedings, 37, 489–490.CrossRefPubMedGoogle Scholar
- 16.Schroder, C., Pfeiffer, S., Wu, G., Zorn, G. L. 3rd, Ding, L., Allen, C., et al. (2003). Effect of complement fragment 1 esterase inhibition on survival of human decay-accelerating factor pig lungs perfused with human blood. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation, 22, 1365–1375.CrossRefGoogle Scholar
- 17.Burdorf, L., Stoddard, T., Zhang, T., Rybak, E., Riner, A., Avon, C., et al. (2014). Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14, 1084–1095.CrossRefGoogle Scholar
- 19.Westall, G. P., Levvey, B. J., Salvaris, E., Gooi, J., Marasco, S., Rosenfeldt, F., et al. (2013). Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation. The Journal of Heart and Lung Transplantation: The official Publication of the International Society for Heart Transplantation, 32, 1123–1130.CrossRefGoogle Scholar
- 20.Daggett, C. W., Yeatman, M., Lodge, A. J., Chen, E. P., Van Trigt, P., Byrne, G. W., et al. (1997). Swine lungs expressing human complement-regulatory proteins are protected against acute pulmonary dysfunction in a human plasma perfusion model. The Journal of Thoracic and Cardiovascular Surgery, 113, 390–398.CrossRefPubMedGoogle Scholar
- 21.Kulick, D. M., Salerno, C. T., Dalmasso, A. P., Park, S. J., Paz, M. G., Fodor, W. L., et al. (2000). Transgenic swine lungs expressing human CD59 are protected from injury in a pig-to-human model of xenotransplantation. The Journal of Thoracic and Cardiovascular Surgery, 119, 690–699.CrossRefPubMedGoogle Scholar
- 22.Rosengard, A. M., Cary, N. R., Langford, G. A., Tucker, A. W., Wallwork, J., & White, D. J. (1995). Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection. Transplantation, 59, 1325–1333.CrossRefPubMedGoogle Scholar
- 23.Rosengard, A. M., Cary, N., Horsley, J., Belcher, C., Langford, G., Cozzi, E., et al. (1995). Endothelial expression of human decay accelerating factor in transgenic pig tissue: A potential approach for human complement inactivation in discordant xenografts. Transplantation Proceedings, 27, 326PubMedGoogle Scholar
- 24.Ramirez, P., Montoya, M. J., Rios, A., Garcia Palenciano, C., Majado, M., Chavez, R., et al. (2005). Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation from pig to baboon using polytransgenic pig livers (CD55, CD59, and H-transferase). Transplantation Proceedings, 37, 4103–4106.CrossRefPubMedGoogle Scholar
- 27.Le Bas-Bernardet, S., Tillou, X., Poirier, N., Dilek, N., Chatelais, M., Devalliere, J., et al. (2011). Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons. Transplantation Proceedings, 43, 3426–3430.CrossRefPubMedGoogle Scholar
- 28.Jeong, Y.-H., Park, C.-H., Jang, G.-H., Jeong, Y.-I., Hwang, I.-S., Jeong, Y., et al. (2013). Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes. PLoS ONE, 8, e63241.CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Azimzadeh, A. M., Kelishadi, S. S., Ezzelarab, M. B., Singh, A. K., Stoddard, T., Iwase, H., et al. (2015). Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein. Xenotransplantation, 22, 310–316.CrossRefPubMedPubMedCentralGoogle Scholar
- 38.Harris, D. G., Quinn, K. J., French, B. M., Schwartz, E., Kang, E., Dahi, S., et al. (2015). Meta-analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood. Xenotransplantation, 22, 102–111.CrossRefPubMedGoogle Scholar
- 39.Chen, Y., Stewart, J. M., Gunthart, M., Hawthorne, W. J., Salvaris, E. J., O’Connell, P. J., et al. (2014). Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors. Xenotransplantation, 21, 244–253.CrossRefPubMedPubMedCentralGoogle Scholar
- 45.Eszterhas, S. K., Bouhassira, E. E., Martin, D. I., & Fiering, S. (2002). Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position. Molecular and Cellular Biology, 22, 469–479.CrossRefPubMedPubMedCentralGoogle Scholar
- 49.Bottino, R., Wijkstrom, M., van der Windt, D. J., Hara, H., Ezzelarab, M., Murase, N., et al. (2014). Pig-to-monkey islet xenotransplantation using multi-transgenic pigs. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14, 2275–2287.CrossRefGoogle Scholar