Generation Of Site-specific Fat-1 Knock-in Pigs At Rosa26 Locus

The Fat-1 gene from Caenorhabditis elegans encodes a fatty acid desaturase which could convert n-6 polyunsaturated fatty acids(n-6 PUFAs)to n-3 polyunsaturated fatty acids(n-3 PUFAs).The balance of n-6 PUFAs and n-3 PUFAs in the body plays an important role in stabilizing cell membrane function,promoting growth and development,maintaining cytokine and lipoprotein balance,anti cardiovascular and cerebrovascular diseases,anti-inflammatory and anti-cancer.However,there are few sources of n-3 PUFAs in the diet,which cannot meet the needs of the public.The generation of transgenic pigs rich in n-3 PUFAs is one of the effective methods to solve the source of n-3 PUFAs.To date,some Fat-1 transgenic pigs have been generated.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 locus via CRISPR/Cas9 system.The Fat-1 gene knock-in pigs was obtained via somatic cell nuclear transfer.The tail fibroblasts were isolated from Fat-1 knock-in piglets to perform karyotype analysis.The results showed Fat-1 knock-in piglets have normal chromosome numbers.To further confirm that there is no non-specific integration of Fat-1 gene,the genome of pig ear tissue was extracted to perform Southern blot and results indicated a single copy of the Fat-1 gene at the pig Rosa26 locus.Fat-1 knock-in sow was mated with Duroc boars and the F1 generations including 7 piglets were born.The PCR and sequencing results showed that three piglets contained the Fat-1gene at the Rosa26 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 including ALA?DPA and DHA and a a significant decrease of n-6PUFAs including LA and AA,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.