الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Xenotransplantation is hindered clinically by the presence of a xenoreactive antibody barrier (Cascalho and Platt, 2001). The first step toward clinical xenotransplantation using pig organs would be to convert the rejection from a discordant to a concordant form (Taniguchi and Cooper, 1997). Multiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival.
CRISPR/Cas9 system was used to target relevant genes (GGTA1, CMAH and β4GalNT2) as a method for highly efficient editing of multiple genes in primary porcine fibroblasts. Editing efficiencies greater than 84 % were achieved for the knockout of GGTA1, β4GalNT2, and CMAH. Thus, the high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblasts for in vitro studies and the production of animal models.
Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that the transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3’ untranslated region (3’UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. In the present study, hCD47 constructs with and without the modified 3’UTR were knocked into the GGTA1 locus of porcine fetal fibroblasts independently using CRISPR. Flow cytometry of the genetically modified cells was used to analyze hCD47 localization. The endoplasmic reticulum, mitochondrial, autophagy, and oxidative stress markers were examined by gene expression analysis and confocal microscopy. Phagocytosis of the genetically modified cells by human macrophages was measured by flow cytometry, and stimulation of human/NHP primate lymphocytes by the cells was examined using a PBMCs proliferation assay. Cells transfected with the construct lacking the 3’UTR (hCD47(3’UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3’UTR