Animal Molecular Breeding 2024, Vol.14, No.1, 106-118 http://animalscipublisher.com/index.php/amb 110 Figure 2 Deep sequencing analysis of the GGTA1, CMAH, and B4GALNT2 target regions in delivered piglets (Adopted from Tanihara et al., 2021) Image caption: * Blue and red indicate the target sequences and PAM sequences of each gRNA, respectively. Green and yellow indicate inserted and modified sequences, respectively. ** The frequency was defined as the ratio of the number of amplicons to the total read number. *** The mutation rate was defined as the ratio of the total number of mutant amplicons to the total read number. WT, wild-type. Underlining indicates the presence of an inframe mutation (Adopted from Tanihara et al., 2021) 3.2.3 Other modifications to enhance immune compatibility (e.g., CD47, HLA-E) Beyond the knockout of xenoantigen genes, other genetic modifications have been explored to enhance immune compatibility. For instance, the overexpression of human CD47 in pigs has been investigated to inhibit phagocytosis by human macrophages, thereby reducing immune rejection (Fu et al., 2020). Additionally, modifications to the swine leukocyte antigen (SLA) genes, such as the knockout of β2-microglobulin (B2M) and the major histocompatibility complex class II transactivator (CIITA), have been shown to reduce the expression of SLA class I and class II molecules, respectively. This reduction in SLA expression decreases the activation of human T cells and prolongs the survival of pig xenografts in human recipients (Hein et al., 2019; Xu et al., 2022). In summary, the use of advanced genetic engineering techniques like CRISPR/Cas9 has enabled the precise modification of specific genes in pigs to reduce xenoantigen expression and enhance immune compatibility, thereby improving the prospects of successful xenotransplantation. 4 Case Studies and Experimental Results 4.1 Notable experiments and their outcomes in reducing rejection Several notable experiments have demonstrated significant progress in reducing xenograft rejection through genetic modifications in pigs. One such study involved the generation of GGTA1, β2-microglobulin (β2M), and CIITA triple knockout (GBC-3KO) pigs using CRISPR/Cas9 technology (Figure 3). This genetic modification effectively reduced hyperacute xenograft rejection and prolonged the survival of pig skin grafts in immunocompetent mice (Fu et al., 2020). Another experiment transplanted kidneys from genetically modified pigs into brain-dead human recipients. The kidneys began to produce urine almost immediately after reperfusion, and no signs of hyperacute or antibody-mediated rejection were observed over a 54-hour study period (Montgomery et al., 2022). These experiments highlight the potential of genetic modifications to mitigate immune responses and improve xenograft survival. Fu et al. (2020) describes the creation of GGTA1−/−β2M−/−CIITA−/− triple gene knockout (GBC-3KO) pigs using CRISPR/Cas9 technology. Guide RNAs targeted exon 8 of GGTA1, exon 2 of β2M, and exon 9 of CIITA. The process involved transfecting pig embryonic fibroblasts (PEFs) with Cas9 and sgRNA vectors, followed by single-cell sorting and genotyping. Successful knockout cell lines underwent somatic cell nuclear transfer, leading to embryo implantation in surrogates. Of the 1 346 transferred embryos, five pregnancies resulted in two natural deliveries, producing five male piglets. Genotyping confirmed the knockout mutations. This research demonstrates effective genetic editing for creating multi-gene knockout pigs for potential xenotransplantation applications.
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