Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5.

Xiaoqing Zhou; Yu Liu; Chengcheng Tang; Lingyin Cheng; Shuwen Zheng; Yuling Zheng; Min Chen; Huaqiang Yang; Qingjian Zou; Liangxue Lai

Return

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5.

Author: Xiaoqing ZHOU ¹ ; Yu LIU ¹ ; Chengcheng TANG ¹ ; Lingyin CHENG ¹ ; Shuwen ZHENG ¹ ; Yuling ZHENG ¹ ; Min CHEN ¹ ; Huaqiang YANG ² ; Qingjian ZOU ¹ ; Liangxue LAI ¹
Author Information

1. School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, Guangdong, China.
2. College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.
Publication Type:Journal Article
Keywords: GGTA1; HLA-G5; TALEN; gene knockout; gene-modified pig; xenotransplantation
MeSH: Animals; Animals, Genetically Modified; Gene Knockout Techniques; HLA Antigens; Humans; Nuclear Transfer Techniques; Swine; Transplantation, Heterologous
From: Chinese Journal of Biotechnology 2022;38(3):1096-1111
CountryChina
Language:Chinese
Abstract: Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.