Production Of GGTA1Deficient Wuzhishan Miniature Pigs

Xenotransplantation is a promising solution to the shortage of human donor organs in transplantations, and pigs are considered to be suitable animals for xenografts. However, organ transplantation from pig to non-human primates results in a series of immunological rejections to the xenografts, in which hyperacute rejection (HAR) is the primary immunological obstacle. HAR is initiated mainly by the binding of the natural antibody named anti-Gal al,3Gal (aGal) preexisting antibodies and complements after transplantation within minutes. The Ga1β1-4G1cNAcα1-3-galactosyltransferase synthesizes the aGal epitopes onto cells surface as a xenoantigen, thus knockout of al,3-galactosyltransferase (GGTA1) gene could avoid HAR by eliminating the aGal epitopes.This study was based on the previously production of single allele knock-out of GGTA1gene in Wuzhishan miniature pigs (WZSP). WZSP fibroblasts with inactivated GGTAl gene were successfully selected by optimized cells screening methods and then utilized for somatic nuclear transfer (SCNT). Approximately4months later, GGTA1gene deficient piglets were obtained for the first time in China. Summary of the study is as follows:Study One:The knockout vector targeting GGTA1gene, pBS-GGTA1-puro, which was based on homologous recombination (HR) and contained a second antibiotic-resistance, puro gene as a marker was utilized to knockout the second allele of the GGTAl gene.36cell colonies were picked up; however, no GGTA1-/-cell colonies were identified by polymerase chain reaction (PCR).Study Two:A kind of spontaneous mutation has been identified in mammalians cells with a heterozygous allele gene, including pigs. As applications of this theory in a few researches have led to successes in production of GGTA1null pigs, several experiments were carried out in this part to select GGTA1-/-cells in fibroblasts derived from WZSP fetuses, by using magnetic cell sorting(MACS), fluorescence-activated cell sorting (FACS) and reagents which bound to Gal al,3Gal epitopes specifically, such as phytolectin GSI-B4and Clostridium difficile Toxin A (TcdA). Cell colonies obtained were analyzed by PCR.(1) After treatments of biotinylated GSI-B4lectin and then streptavidin-conjugated magnetic beads, the cells that were not captured with lectin and magnetic beads were subcultured in new dishes. Eleven colonies were picked up and two of which were identified as GGTA1-/-cells. The rest cells that couldn’t be separated were expanded for FACS screening, and three mixed cell collections were screened out and none of them were GGTA1-/-cells as expected.(2) Four colonies were collected by FACS followed by TcdA treatment, and all of them were GGTA1-/-cells. To sum up, a total of eighteen colonies were gained in these experiments and six of which were affirmed as GGTA1-/-cell colonies by PCR, and the total efficiency was33.3%.Study Three:To generate GGTA1-/-piglets, oocytes from sow ovaries were matured in vitro, and NT was performed. Totally3122embryos were transferred into twelve sow recipients, and four pregnancies went to term and delivered twelve live born piglets. Fibroblasts derived from ear explants of the twelve piglets were established for subsequent experiments. Eleven piglets were verified as GGTA1gene deficient piglets by PCR, Southern blotting. The ear fibroblasts from the eleven piglets seperately stained with FITC-labeled GSI-B4lectin and monitored by flow cytometry revealed almost no aGal epitopes expressed on these cells surfaces.The above results suggested that the difficulties of second-round of GGTA1gene targeting were beyond our imagination for its extremely low efficiency. Nevertheless, cells lacking of aGal epitopes brought by the spontaneous mutations in WZSP fibroblasts could be isolated through certain methods based on phenotype screening. Moreover, proper combinations of screening methods could bring higher efficiency.The first production of GGTA1gene deficient WZSP in China has laid a solid root for further study of genetic modification in pigs for xenotransplantation.