Production And Characterization Of Fat1 And Fat2 Gene Fusion Expression Transgenic Pigs And Co-expression Transgenic Mice

Polyunsaturated fatty acids(PUFAs)are a kind of long chain fatty acid containing poly-unsaturated bonds.According to the numbers and positions of unsaturated bonds,PUFAs can be divided into n-3 and n-6 PUFAs.It can promote the neural development and fetal growth in mammals and prevent the risk of cardiovascular disease in humans.PUFAs are the essential fatty acid for human health,which must be absorbed from diet.In China,pork is the main meat source in Chinese food.Aiming to increase the PUFAs content in pork is of great significance to improve the level of people's health.In order to solve the problem of insufficient PUFAs content in pork,we cloned the genes of two enzymes,delta-12-fatty acids desaturation gene(FAT2)and delta-15-fatty acids desaturation gene(FAT1),which play key roles in PUFAs synthetic pathway.We then produced the fat1-fat2 fusion expression transgenic pigs and fat2-2A-fat1 co-expression transgenic mice.This two animal models had increased PUFAs content in muscle and fat tissues.This study provides the basics research on producing the endogenous PUFAs transgenic animal.Trial 1: Production and characterization of fat1-fat2 fusion expression transgenic pigsTo produce the transgenic pigs,the p CAG-fat1-fat2 fusion expression vector transfected into pig's fetal fibroblast cells and transferred to recipient sows by somatic cell nuclear transfer method(SCNT).In total,5 transgenic pigs were born,and the transgenic pigs had stable expression of transgenic protein in 3 and 120 days old.The results of q PCR and RTPCR showed that the fat1-fat2 fusion gene at the peak expression in transgenic pigs' muscle tissues.The n-3 PUFAs content increased by 30.34% in 3 days old transgenic pigs' muscle tissue,and n-6 PUFAs increased by 32.64%.For the 120 days old transgenic pigs,the n-3 PUFAs content of muscle,fat,skin tissues increased by 41.84%(p<0.01),25.23%(p<0.01),22.90%(p=0.05),respectively,and n-6 PUFAs decreased by 25.01%(p<0.05),24.32%(p<0.01),36.17%(p=0.01),respectively.The expression of NF?B,SREBP1 c and FAS genes were down-regulated in transgenic pig's three tissues;the expression of PPAR?,ELOVL2 and ELOVL5 genes were upregulated in those tissues;but the expression of LXR? gene had no effect in those tissues.Trial 2: The construction of fat2 and fat1 co-expression plasmid,expression in cells and functional verification.In order to improve the expression efficiency of fat1 and fat2 gene,we used the overlap extension PCR to construct the self-cleavage and co-expression gene linked by 2A sequence: fat2-2A-fat1 gene,then we assembled this gene to the co-expression plasmid: p CAG-fat2-2A-fat1-EGFP.The plasmid transfected into PK15 cells,and the fat2-2A-fat1 m RNA can be detected in cells after 48 hours.Detection of PUFAs content in transfection cells showed that the n-3 PUFAs content increased by 30.7%(P=0.05),and n-6 PUFAs decreased by 34.74%.Trial 3: Production and characterization of fat2 and fat1 co-expressing transgenic miceWe used the co-expression plasmid(constructed from trial 2)to produce the transgenic mice by pronuclear microinjection.In total,6 F0 transgenic mice were born.The results of PCR,Southern blot,m RNA expression and the PUFAs content in muscle tissue trials showed that the 606 transgenic mice had the highest gene expression in F0 mice.As male parent,606 mice produced 11 transgenic mice(F1)and 26 transgenic mice(F2).The n-6 PUFAs content of F0 transgenic mice in muscle tissue increased by 30.61%(p<0.01),and n-3 PUFAs increased by 6.87%.For the fat tissue,n-6 PUFAs decreased by 29.47%(p<0.01),n-3 PUFAs decreased by 29.20%(p<0.01).For skin tissue,n-6 PUFAs increased by 8.39%,n-3 PUFAs decreased by 33.10%.The n-6 PUFAs content of F1 male transgenic mice in muscle tissue increased by 4.31%,n-3 PUFAs increased by 9.47%.The n-3 PUFAs content of F1 female mice increased by 16.34%,n-6 PUFAs increased by 22.64%.For the fat tissue,the n-3 PUFAs content of the male mice increased by 7.21%,n-6 PUFAs increased by 6.50%.The n-6 PUFAs content of female mice increased by 16.50%,n-3 PUFAs decreased by 5.93%.Transgenic mice had different expression levels of fat2 and fat1 genes and PUFAs contents may be effected by different tissues and genders.The expression of FAS,SREBP1 c and LXR? genes were down-regulated in transgenic mice parts of tissues.The expression of PPAR?,ELOVL2 and ELOVL5 genes were upregulated in three tissues.The expression of NF?B gene was not affected in those three tissues.In conclusion,we successfully produced the fat1 and fat2 gene fusion expression transgenic pigs and co-expression transgenic mice.These two transgenic animals expressed fat1 and fat2 gene,and improved the PUFAs content in animals.This study provided a new solution to produce a new varieties of transgenic livestock rich in polyunsaturated fatty acid in future.