Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs

Abstract

The fat-1 gene from Caenorhabditis elegans encodes a fatty acid desaturase which was widely studied due to its beneficial function of converting n-6 polyunsaturated fatty acids (n-6PUFAs) to n-3 polyunsaturated fatty acids (n-3PUFAs). To date, many fat-1 transgenic animals have been generated to study disease pathogenesis or improve meat quality. However, all of them were generated using a random integration method with variable transgene expression levels and the introduction of selectable marker genes often raise biosafety concern. To this end, we aimed to generate marker-free fat-1 transgenic pigs in a site-specific manner. The Rosa26 locus, first found in mouse embryonic stem cells, has become one of the most common sites for inserting transgenes due to its safe and ubiquitous expression. In our study, the fat-1 gene was inserted into porcine Rosa 26 (pRosa26) locus via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. The Southern blot analysis of our knock-in pigs indicated a single copy of the fat-1 gene at the pRosa26 locus. Furthermore, this single-copy fat-1 gene supported satisfactory expression in a variety of tissues in F1 generation pigs. Importantly, the gas chromatography analysis indicated that these fat-1 knock-in pigs exhibited a significant increase in the level of n-3PUFAs, leading to an obvious decrease in the n-6PUFAs/n-3PUFAs ratio from 9.36 to 2.12 (***P < 0.0001). Altogether, our fat-1 knock-in pigs hold great promise for improving the nutritional value of pork and serving as an animal model to investigate therapeutic effects of n-3PUFAs on various diseases.

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication

“…CRISPR/Cas9 system has been widely used in generation of gene modification cell lines or animals. In the transgenic research in pigs, a number of knock-in or knockout porcine cells and pigs have been generated using CRISPR/Cas9 system [ 30 – 33 ]. Such as the critical region of fusion receptor of porcine reproductive and respiratory syndrome virus (PRRSV), CD163 subdomain 5 has been knocked out from porcine immune cells.…”

Reflections on the system of evaluation of gene-edited livestock

“…This provides more genotype for selection combined with somatic cell nuclear transfer, and the use of GE has greatly increased the probability of obtaining positive individuals following microinjection of one-cell embryos. In recent years the species of gene-edited livestock (a new breeding material) have been steadily increasing [13][14][15][16][17][18][19][20][21][22] . This great genetic progress has also led to social concerns [23] .…”

Livestock 2.0 – genome editing for fitter, healthier, and more productive farmed animals

“…For example, Lai and colleagues generated cloned pigs that expressed the fat-1 gene from the nematode Caenorhabditis elegans and that exhibited significantly reduced ratios of n-6 to n-3 fatty acids, which might have human health benefits [ 39 ]. Although some have questioned the value of such pigs [ 40 ], nevertheless others have also generated pigs carrying the C. elegans fat-1 gene (which encodes an n-3 fatty acid desaturase) and have observed similar changes [ 41 , 42 ], including Li and colleagues, who used ‘clustered regularly interspaced short palindromic repeats’ (CRISPR)–CRISPR-associated 9 (Cas9) gene-editing technology for the directed integration of the fat-1 gene from C. elegans into the porcine Rosa 26 locus [ 43 ].…”