International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 161 generating multi-gene-modified pigs, which can significantly enhance the efficiency and success rate of pig-to-human xenotransplantation research. Figure 4 Immunohistochemical assessment of wild-type and GGTA1/CMAH double-edited piglets (Adopted from Tanihara et al., 2021) Image caption: Ear biopsies derived from wild-type (WT) and GGTA1/CMAH double-edited piglets (#2 and #3) were immunohistochemically stained for Galα(1,3)Gal (green) (a) and Neu5Gc (green) (b). These tissues were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (blue); The scale bar in each panel represents 50 μm (Adopted from Tanihara et al., 2021) Figure 4 presents the analysis results of ear biopsy samples from wild-type and GGTA1/CMAH double-gene-edited pigs using the immunohistochemistry method. The samples were stained with Alexa 488-labeled heterologous lectin B4 and anti-Neu5Gc antibodies to detect the expression of Galα(1,3)Gal and Neu5Gc epitopes. The results show that in the GGTA1/CMAH double-gene-edited pigs, the absence of Galα(1,3)Gal is clearly visible in Figure 3a, while the expression of Neu5Gc epitopes is similar to that of the wild-type, as shown in Figure 3b. This indicates that despite gene editing, some xenoantigens are still expressed, demonstrating the variability in the effects of gene editing and the presence of mosaicism. Another study highlighted the use of CRISPR/Cas9 to edit patient-derived xenografts, showcasing the technology's ability to perform rapid in vivo functional genomics and analyze genetic dependencies and drug resistance mechanisms (Hulton et al., 2020). These case studies underscore the transformative impact of CRISPR/Cas9 on the field of xenotransplantation, paving the way for more effective and safer transplantation practices. By leveraging the precision and efficiency of CRISPR/Cas9, researchers are making significant strides in overcoming the challenges associated with xenotransplantation, ultimately bringing us closer to the goal of using animal organs to address the shortage of human donor organs. 5 Ethical and Regulatory Considerations 5.1 Ethical concerns surrounding genetic modification and xenotransplantation The application of CRISPR/Cas9 technology in xenotransplantation raises significant ethical concerns. One primary issue is the moral implications of genetic modification in animals, particularly pigs, which are commonly used as organ donors. The genetic alteration of these animals to reduce immunogenicity and improve compatibility with human recipients involves complex ethical considerations regarding animal welfare and the extent to which humans should interfere with natural genetic processes (Ryczek et al., 2021; Kararoudi et al., 2018). Additionally, the potential for unintended off-target effects and genetic mosaicism in edited animals poses further ethical dilemmas, as these could lead to unforeseen health issues in the animals or compromise the safety of the transplanted organs (Tanihara et al., 2021; Zhang, 2020). The broader ethical debate also encompasses the potential long-term impacts on biodiversity and the natural ecosystem, as well as the moral status of genetically modified organisms (Zhang et al., 2020). 5.2 Regulatory frameworks governing the use of CRISPR/Cas9 in xenotransplantation The regulatory landscape for the use of CRISPR/Cas9 in xenotransplantation is still evolving. Different countries have established varying frameworks to address the safety, efficacy, and ethical implications of this technology.
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