Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.
Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
Kimyung
K
Choi
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
Joohyun
J
Shim
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
Nayoung
N
Ko
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
Heejong
H
Eom
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
Jiho
J
Kim
Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Jeong-Woong
JW
Lee
Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
Dong-Il
DI
Jin
Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea. hikim@optipharm.co.kr.
Hyunil
H
Kim
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015703:Antigens, CD / Q000235:genetics*; D001081:Apyrase / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques; D006579:Heterozygote; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D013552:Swine; D013556:Swine, Miniature / Q000235:genetics; D014183:Transplantation, Heterologous
D015703:Antigens, CD; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; D001081:Apyrase; C067869:CD39 antigen
Human CD39; Pig; Transgenic; Xenotransplantation; α1,3-Galactosyltransferase