Department Physiology, University of Murcia, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum" and Institute for Biomedical Research of Murcia (IMIB-Arrixaca), 30100, Murcia, Spain.
IIS Biodonostia, Neuroscience, San Sebastián, Spain.
Theriogenology. 2024 Apr 1;218:111-118. doi: 10.1016/j.theriogenology.2024.01.040. Epub 2024 Feb 1.
Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.
基因修饰猪在模拟人类疾病、异种移植以及开发对病毒疾病具有抗性的猪方面发挥着关键作用。可编程内切酶的应用，包括 CRISPR/Cas9 系统，彻底改变了基因修饰猪的产生方式。本研究评估了在受精前对卵母细胞进行电穿孔以产生不同模型编辑基因胚胎的效率。对于单基因编辑，使用了磷脂酶 C ζ (PLC ζ) 和融合肉瘤 (FUS) 基因，并优化了 sgRNA 和 Cas9 复合物的浓度。结果表明，增加浓度会导致更高的突变率，而不会影响囊胚率。电穿孔可高效产生 TPC1/TPC2 基因的双敲除（79%）。此外，通过电穿孔还实现了对 PRRS 和猪流感等病毒疾病的抗性，从而产生了双敲除胚胎猪（63%）。该研究还证明了使用电穿孔在单个步骤中进行多个基因编辑的潜力，这与异种移植相关。该技术可同时突变 5 个基因（GGTA1、B4GALNT2、假 B4GALNT2、CMAH 和 GHR）。总体而言，电穿孔被证明是一种高效且多功能的方法，可用于产生基因修饰的胚胎猪，在生物医学和农业研究、异种移植和疾病抗性方面取得了重大进展。电穿孔可在单个会话中处理大量卵母细胞，同时使用更便宜的设备。我们证实了在不改变胚胎发育到囊胚阶段的情况下，同时对单个、双个或五个基因进行基因编辑的猪胚胎的产生。这些结果为不同模型的基因编辑方案的优化提供了有价值的见解，为该领域的研究和应用开辟了新的途径。
