The Detection And Verification In Transgenic Mice And Cloned Pig Of Muscle-Specific Overexpression Swine Dgat1Gene

Pork is the important source of human intake of animal protein. High quality pork performs is tender and juicy. Intramuscular fat (IMF) has a very high correlation with those important economic traits and becomes the significant index for assessing pork quality. In last century, the target of pig breeding was increasing lean meat content and reducing backfat thickness, which caused dramatic decrease of IMF content (dropped to1.5%, recommended range:2.5-3%) and had negative influence on pork palatability and quality. In future, without reducing the lean meat content, the goal of swine breeding has been changed into increasing IMF content which means the augmentation of triglyceride (TAG) content. DGAT1is not only the rate-limiting enzyme which catalyzes the final step of triglyceride synthesis, but also the important role that involved in the regulation of mammalian lipid metabolism. Therefore, we took DGAT1as a candidate gene and constructed the eukaryotic expression vector sMCK-sDGAT1. We obtained transgenic mice and pigs by micro injection and somatic cell cloning, to study the effect of muscle-specific overexpression of DGAT1gene. The results are as follows:1. We arrested swine MCK gene’s promoter sequence from BAC (bacterial artificial chromosome) by recombinant DNA technology. Eukaryotic expression vector pEGFP-N1(as a framework) and the synthesized full-length swine DGAT1coding sequence were used to construct muscle-specific expression vector sMCK-sDGAT1. The vector sMCK-sDGATl was correct which proved by polymerase chain resction、restriction enzyme assay and sequence analysis.2.16positive transgenic Fouder mice were made in Shanghai Bio model Technology Co., Ltd by pronuclei microinjection. We obtained F1generation (positive male mice mating with negative C57BL/6J female mice). The results of real time PCR show that DGAT1gene of transgenic mice is highly expressed in skeletal muscle and kidney, medium expressed in cardiac muscle and rarely expressed in the brain, lung, intestine and other tissues. Those results are consistent with the expression in various tissues of mammalian MCK gene.3. We detect genes expression which involved in fat synthesis and transport of skeletal muscle by real-time PCR. The results showed that related gene expression had increased, such as the plastid acetyl-CoA carboxylase1increased34%(p<0.01), fatty acid binding protein (53%, p<0.05), fatty acid synthase (74%, p<0.01), the ethylene precursor ACC synthase (62%, p<0.05) and the brown fat-specific genen uncoupling protein (74%, p<0.01). Western Blot improved that protein level of DGAT1、ACC1、FABP4and UCP1had increased. The difference of weight between positive mice and negative mice was not significant by continuous determination, indicating the increase of IMF will not cause the mouse obesity. Determination of triglycerides and fatty acids in skeletal muscle showed the DGAT1positive mice increased35.5%(p<0.01) and20.3%(p<0.01), respectively.4. We cultured porcine primary fibroblast cell lines. Then vector sMCK-sDGAT1was linearized and stably transfected into fibroblast by electric shock. We obtained positive monoclonal cells after G418selection using PCR assay. Finally, we obtained transgenic pig by somatic cell nuclear transfer.5. PCR and Southern Blot were used to identify the founder transgenic pigs and one founder transgenic pig was detected. Absolute quantitative PCR was employed to calculate the transgene copy number. The parameters of the standard curve was:Log2N=-0.6497△Ct+4.9372(R2=0.9835, p<0.01), and copy number is32.893±1.99.