Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes

Abstract

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT−(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-016-9979-8) contains supplementary material, which is available to authorized users.

Strong xenoprotective function by single‐copy transgenes placed sequentially at a permissive locus

“…[26][27][28] Obtaining sufficient and ubiquitous CD55 expression has however been difficult. 9,29 Good results have been gained with pigs carrying multiple copies of CD55 minigenes, 30,31 and CD55 expression equivalent or higher than the corresponding human tissue obtained using a porcine CD46 promoter. 31 Our CD55 minigene is similar, but uses the CAG promoter.…”

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73

“…The oocytes were matured for 40 h at 38.5℃ under 5% CO 2 in air. Somatic cell nuclear transfer was performed as follows ( Kwon et al, 2017 ). Briefly, matured oocytes were enucleated by aspirating the first polar body and metaphase II chromosomes and a small amount of surrounding cytoplasm in manipulation medium supplemented with 5 µg/mL cytochalasin B.…”

The Landscape of Genomic Imprinting at the Porcine SGCE/PEG10 Locus from Methylome and Transcriptome of Parthenogenetic Embryos

“…Pig oocytes were collected and in vitro maturation (IVM) was performed as described in our previous reports (Kwon et al 2017;Ahn et al 2020). In brief, from a local slaughterhouse (Nonghyup Moguchon, Gimje, Korea), ovaries from Landrace x Yorkshire x Duroc (LYD) pigs were obtained, and then immediately transported to our laboratory in saline solution supplemented with penicillin and maintained at 30-35°C in a thermos.…”