Construction Of Swine Muscle-specific Overexpression PPARγ2Eukaryotic-Epressing Vectors And The Function Verification In Transgenic Mice

Intramuscular fat (Intramuscularfat, the IMF) is material basis of the meat tender and juicy and meat flavour, for increasing the content of the IMF can make the muscle tenderness, juiciness and flesh increased. the intramuscular fat content has become the most critical parameters of meat quality indicators in the Assessment of pork quality indicators. However, since the1970s, Domestic and foreign experts has focused on the choice for lean meat increased in the lean pig breeding, the high lean meat choices lean pigs IMF content dropped by about1.5%, lower than the optimum range, which greatly reduced pork quality. How can we improve the IMF under the premise to ensure lean meat content has become a new target for breeder. Evidence emerging from studies of humans and mice has indicated peroxisome proliferator-activated receptor y (PPARy) to be not only a key factor for adipogenesis but also a critical determinant of body fat distribution. Therefore, we use PPARy2as a candidate gene in this study, to investigate the consequences of swine PPARy2(sPPARy2) overexpression vector and the influence on fat in mouse skeletal muscles, we construct muscle-specific eukaryotic expression vector using a7-kb swine muscle creatine kinase (sMCK) promoter to drive sPPARy2into ectopic expression in a transgenic (TG) mouse model for the first time. Following are the important results.We used Eukaryotic expression vector pEGFP-N1as a framework, arrested from a BAC of swine MCK gene by gene technology, obtained7Kb MCK promoter sequence to construct the vector pEGFP-N1L-MCK. The full-length sPPARy2CDS (SaiⅠ and NotⅠ restriction sites were introduced at the5'-and3'-ends) was synthesized and subcloned into the pMD18-T vector. The pMD18-T vector and the pEGFP-N1L-sMCK vectors were digested with SalⅠ/NotⅠ enzymes to replace the EGFP sequence with the sPPARy2sequence. The final vector pEGFP-N1L-sMCK-sPPARy2was sequenced and linearized by SwaⅠ and used for pronuclear injection.The results of part sequencing, PCR assay, and BglⅡ restriction enzyme analysis are correct, meaning that the Eukaryotic expression vector pEGFP-N1L-sMCK-sPPARy2was successfully constructed.We cultured primary fibroblast cell lines using pig35-day-old embryos, And transient transfected fibroblast cell with the eukaryotic expression vector pEGFP-N1L-MCK-PPARγ2. The results of RT-PCR analysis showed that mRNA of PPARγ2in fibroblasts was overexpression that upregulated the expression of fat gene SCD2, LPL and ACC. At the same time,we transfected the linearized vector in mouse myoblasts C2C12and selected with G418, acquired the stably transfected cell lines, The results of RT-PCR analysis showed that expression of PPARγ2-positive cells increased many times compared with negative cells. Through the above two transfection experiments can be seen that MCK promoter could drive the PPARγ2overexpression at the cellular level.We obtained5positive founder mice by microinjection. Respectively, positive male and female mice with negative heterosexual mice mating, giving birth to the F1generation, the2week-old mice were identified by polymerase chain reaction (PCR)-based genotypingusing. We detected the two months old Fl generation mice using RT-PCR and Western Blot. The results of RT-PCR showed PPARγ2is highly expressed in skeletal and cardiac muscle of transgenic mice, while expression level are rarely in the liver, kidneys, medium, fat, small intestine, testis. This result matched the expression of MCK in mammalian tissues, indicating that the eukaryotic expression vector in transgenic mice could express normally and also showing that the pig MCK promoter can drive gene overexpressing in the muscle tissue like the mouse MCK promoter.To verify the effect of the overexpression of sPPARγ2in skeletal muscle, we detected several target genes or adipocyte genes by real-time PCR. In contrast to WT mice, some genes were up-regulated in TG mice, including fatty acid-binding protein4(FABP4; P<0.01), antigen (CD36)(CD36; P<0.01), fatty acid synthase (FAS; P<0.05), lipoprotein lipase (LPL; P<0.01), and diacylglycerol O-acyltransferase1(DGAT1; P<0.05), whereas stearoyl-Coenzyme A desaturase2(SCD2) was down-regulated to about40%of that of WT mice (P<0.01). Hormone-sensitive lipase (HSL), a key gene that degrades triacylglycerol, was decreased62%(P<0.01).Continuous determination of mice4-16weeks of weight showed that the difference of weight between positive mice and negative mice was not significant. skeletal muscle triglyceride increase of30%(p<0.01), free fatty acids increased16%(p<0.01), while blood triglyceride and plasma glucose were no significant differences. A detailed analysis of single fatty acids in skeletal muscle by gas chromatograph indicated obvious increase in palmitoleic (16:1)(P<0.01), linoleic (18:2)(P<0.01), arachidonic (20:4) and docosahexaenoic (22:6) acids(P<0.01), while myristic (14:0) and oleic (18:1) acid (P <0.01) were decreasedExperiments on skeletal muscle glucose metabolism related genes were detected and found that the mRNA expression of glucose transporter the protein1(Glutl), glucose transporter the protein4(Glut4), phosphate glucose isomerase1, fructose bisphosphatase2, pyruvate dehydrogenase kinase isoenzyme1was increased, while phosphate phosphoglycerate mutase1was decreased, the expression of glycogen synthase2was not change significant.In the trial, we detect some genes only express in brown fat by RNA and protein synthesis, including uncoupling protein UCP1, which is a brown-fat-specific marker gene, PRDM16, PGC1, Cidea,Cox7a. Result of Real Time PCR show that UCP1was increased remarkablly both in mRNA and protein level in subcutaneous fat of TG mice (p<0.01). Other brown fat gene PRDM16also increased by79%(P<0.01), the other PGC1α (2.2fold, P<0.01), Cidea (21fold, P<0.01), Cox7a (7.4fold, P<0.01) increased in varying degrees, and reach a significant level.We detected the expression of fat-related genes in liver of mice, results showed that the mRNA of PPARy2increase more than8times, of FAS to increase210-fold, of DGAT1increase the93%, while HSL to reduce to13%, and FABP4increase to2.2-fold, explain the liver synthesized more of the fatty acids and triglycerides were diverted to other organizations, The results of liver tissue sections HE staining shown that the difference of fat in the liver was not significant.