| Summary: | Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (<i>Fat-1</i>) and porcine insulin-like growth factor-1 (<i>IGF-1</i>) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the p<i>Rosa26</i> locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the <i>Fat-1</i> and <i>IGF-1</i> genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** <i>p</i> < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.
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