International Journal of Molecular Zoology 2024, Vol.14, No.4, 244-254 http://animalscipublisher.com/index.php/ijmz 246 compared to CRISPR/Cas9. But it is more complex and time-consuming to design and produce, which can limit their widespread use. ZFNs has long history of use and well-understood mechanisms, while has lower efficiency and higher cost compared to CRISPR/Cas9 and TALENs. 3.3 Specific applications of genetic engineering in creating pathogen-free pigs Genetic engineering has been pivotal in creating pathogen-free pigs for safer xenotransplantation. Some specific applications include: Inactivation of PERVs: Using CRISPR/Cas9, researchers have successfully inactivated PERVs in the pig genome, reducing the risk of viral transmission during xenotransplantation (Kemter et al., 2018; Yue et al., 2020). Elimination of Xenoantigens: Genetic modifications have been made to eliminate specific carbohydrate antigens such as αGal, Neu5Gc, and Sd(a), which are targets for human antibodies. This reduces the risk of acute rejection of xenotransplants. Expression of Human Transgenes: Pigs have been engineered to express human complement regulatory proteins (e.g., hCD46, hCD55, hCD59) and other transgenes that enhance immunological compatibility and reduce coagulation-related issues during xenotransplantation (Yue et al., 2020). Improvement of Islet Function: Genetic modifications, such as the expression of glucagon-like peptide-1 and M3 muscarinic receptors, have been shown to increase insulin secretion in porcine islets, potentially improving the outcomes of islet xenotransplantation for diabetes treatment (Kemter et al., 2018). In summary, the advancements in genetic engineering technologies, particularly CRISPR/Cas9, have significantly contributed to the development of pathogen-free pigs, enhancing the safety and efficacy of xenotransplantation. These technologies offer promising solutions to overcome immunological barriers and reduce the risk of pathogen transmission, paving the way for clinical applications in the near future. 4 Advances in Creating Pathogen-Free Pigs 4.1. Targeting endogenous retroviruses (e.g., PERVs) using gene editing Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs and pose a significant risk for xenotransplantation due to their potential to infect human cells. Recent advances in gene editing, particularly the use of CRISPR/Cas9, have enabled the inactivation of PERVs in pigs. This approach involves mutating the pol genes of PERVs to prevent their replication and transmission (Figure 1) (Denner, 2021). Studies have shown that PERV-inactivated pigs can be produced, significantly reducing the risk of PERV transmission during xenotransplantation (Kemter et al., 2018; Niu et al., 2020; Denner, 2022). Figure 1 Schematic presentation of the genome of PERV (Adopted from Denner, 2021) Image caption: LTR, long terminal repeat; gag, group specific antigen; pol, polymerase; env, envelope. The recombinant PERV-A/C is the result of a recombination in the env gene spanning the receptor binding domain (RBD). During passaging of PERV-A and PERV-A/C on human cells, a multimerization of repeats in the LTR takes place (Adopted from Denner, 2021) The research of Denner (2021) presents a schematic representation of the genome structure of different types of Porcine Endogenous Retroviruses (PERVs), specifically PERV-A, PERV-C, and their recombinant form
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