CRISPR/Cas9-mediated Site-specific Knock-in DGAT1 Transgenic Pig Development

Intramuscular fat(IMF)content has become one of the most important parameters in the evaluation of meat quality,and has become an important guarantee for pig muscle quality.Increasing the IMF content of the pig can improve the tenderness,moisture and flesh color of the pork.IMF is mainly composed of triglyceride(TAG)and phospholipids.The content of TAG directly affects the content of IMF,thus affecting the meat quality of pork.Diacylglycerol acyltransferase(DGAT1),as an acyltransferase,uses diglyceride and fatty acid as substrates to catalyze the synthesis of TAG,which affects the change of IMF content and affects muscle quality by affecting the change of TAG content.In this study,the LA-CKM-DGAT1-Loxp-Neo-Loxp-RA-TK vector(Donor vector)containing the Neo and TK positive and negative selection marker genes was co-electroporated with the sg RNA-Cas9 vector.Transfer to porcine fetal fibroblasts(PFFs),use G418 and ganciclovir(GCV)positive and negative screening to obtain cell clones,identify positive clones by PCR and DNA sequencing,and remove them by Cre-Loxp recombination system.The Neo-tagged gene was obtained by somatic cell nuclear transfer and embryo transfer to obtain Rosa26 unlabeled transgenic pigs that were knocked into the DGAT1 gene,and the traits of the transgenic pigs were analyzed.The main results obtained are as follows:1.DGAT1 transgenic PFFS cell line was obtained,and DGAT1 transgenic pig(Large white pig)was prepared by somatic cell nuclear transfer and embryo transfer technology.Six transgenic pigs were successfully identified,of which 2 were stillbirths and 1 was unlabeled transgenic pigs.2.Detection of foreign gene copy number in DGAT1 transgenic pigs,the result is a single copy.Tissue expression profiling revealed that DGAT1 was significantly higher in transgenic pig kidneys than in other tissues(p<0.01),and it was significantly up-regulated in muscle tissue.Western blot analysis showed that the expression of DGAT1 in the longissimus dorsi and biceps muscle of transgenic pigs was significantly higher than that of negative pigs(p<0.01).3.In the serum and skeletal muscle physiological and biochemical indicators,it was found that the glycerol triglyceride content in serum and skeletal muscle of DGAT1 transgenic pigs was significantly higher than that of negative pigs(p<0.01).4.The determination of meat quality traits in transgenic pigs after slaughter showed that the IMF content and meat color score of transgenic pigs were significantly higher than that of negative pigs.5.Oil red O staining of the longest muscle tissue section of DGAT1 transgenic pigs showed that the distribution of lipid droplets in the muscle of transgenic pigs was significantly larger than that of negative pigs.6.The detection of the longest intramuscular fat-related gene in transgenic pigs showed that PDK4,ACC1,ADRP,HSL,FABP4 and PPARγ were higher in transgenic pigs than in negative pigs.In summary,this study successfully obtained the label-free transgenic pigs that were knocked into DGAT1 at a fixed point,which laid a foundation for the improvement of pork quality.