The Construction And Phenotypic Analysis Of PEPCK-C^mus Transgenic Tibetan Miniature Pigs

Pork is the main meat of the Chinese people. To ensure people across the country with the adequate, high-quality pork, the meat supply is related to an important event in people’s daily lives. With population growth and the improvement of people’s living standards, pork quality and taste requirements are increased in addition to an increase in demand for pork.Phosphoenolpyruvate carboxylase kinase catalytic gluconeogenesis in a rate-limiting reaction, which convert oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO2 response. There are two kinds of PEPCK cells isozyme, respectively located in the mitochondria(PEPCK-M) and cytoplasmic(PEPCK-C) in. PEPCK-C gene(also called PCK1) encodes a 63 kDa protease, expressed mainly in the liver and renal cortex, involved the catalytic reaction of gluconeogenesis. In the liver, as well as white and brown adipose tissue, PEPCK-C is also involved in gluconeogenesis glycerol reaction. Furthermore, PEPCK-C in cataplerosis plays a role in the citric acid cycle to remove anions.PEPCK-C activity in different tissues is regulated at the transcriptional level. For example, in the liver-specific expression promoter sequence PEPCK-C gene is just from-460 bp to + 69 bp. In addition, nutrients and hormones can also regulate transcription levels of PEPCK-C gene. That cAMP, glucocorticoids and insulin can regulate transcription of PEPCK-C gene have been identified and their corresponding sub-start on the regulatory sequences in the PEPCK-C. The study on transgenic mice showed that promoter sequence from-460 bp to + 69 bp contains the cis-acting element-mediated cAMP and glucocorticoid signals can appropriately regulate promoter activity according to cAMP and glucocorticoid signals.Under normal circumstances, PEPCK-C does not express or express very low in skeletal muscle. The study found that overexpression of PEPCK-C gene in mouse skeletal muscle decline the body fat content of PEPCK-Cmus transgenic mice, which is less than half the body fat content of the control mice, but the transgenic mice compared to the control there is more fat distribution in skeletal muscle of mice. According to changes in muscle and fat content of the PEPCK-Cmus transgenic mice, we expect the PEPCK-Cmus trans fat content in the body of the pig gene decreased, and increased inter-muscular fat content, so the taste of the PEPCK-Cmus transgenic pig meat can greatly increase.Objective: By constructing skeletal muscle-specific expression of PEPCK-C gene in Tibetan miniature pigs,PEPCK-Cmus transgenic pigs alter metabolic pathways of glucose and lipids. In order to change the fat content in skeletal muscle and to improve the taste and quality of pork and obtain high lean, muscular fat content increased between varietiesMethod: 1. Construct muscle-specific transgene expression vector PEPCK-Cmus in pig skeletal, and then build PEPCK-Cmus transgenic fibroblasts of pig: the vector contains a 2kb α-skeletal muscle actin(α-skelatal actin) promoter, 3’untranslated region of the PEPCK-C of bovine growth hormone cDNA gene(bGH), and with a screening of the gene transfer vector resistance gene zeocin. The constructed transgenic pig vector transfected fibroblasts, and then get resistance gene PEPCK-Cmus pigs transgenic fibroblasts.2. Construction of PEPCK-Cmus transgenic pigs: fibroblasts with porcine PEPCK-Cmus transgene as donor cells, into which the nucleus has been removed in porcine oocyte nucleus, in vitro cultured porcine blastocysts obtained, and then the blastocysts were transplanted into the uterus of pseudopregnant females pigs after hCG injection and finally get the PEPCK-C transgenic pigs by somatic cell nuclear transfer.3. The analysis of PEPCK-Cmus transgenic pig genotype and phenotype: detection of PEPCK-Cmus transgenic pig genotypes by PCR; the level of RNA and the protein of PEPCK-C was determined between PEPCK-Cmus transgenic pig skeletal muscle and WT pigs by RT-PCR and Western blot analysis method; the fat content of the amount of each tissue of PEPCK-Cmus transgenic pig skeletal muscle and WT pigs was determined by the method of histology and GC methods.Results: By construction of the skeletal muscle-specific transgene expression vector PEPCK-C in the pig, we get the PEPCK-Cmus transgenic pig fibroblasts; then we get 11 somatic cell nuclear transfer of PEPCK-Cmus transgenic pigs; RT-PCR showed that the PEPCK-C gene expression levels of PEPCK-Cmus transgenic pig muscle increased than the levels PEPCK-C gene of expression of normal porcine muscle; western blot showed that PEPCK-Cmus transgenic pig muscle PEPCK-C expression levels than normal porcine muscle PEPCK-C expression increased; HE staining combined with oil red staining showed increased PEPCK-Cmus transgenic pig muscle fat content than normal porcine muscle fat content; gas chromatographic study shows that the total fat content in the PEPCK-Cmus transgenic pig muscle increase than the fat content of normal porcine muscle.Conclusion: Overexpression of skeletal muscle PEPCK-C gene in pigs, PEPCK-Cmus transgenic pig muscles significantly increases fat content and improves the taste of the pork. It offers a new insight into pig breeding new varieties. At the same time it provides a new animal model for study on the role of PEPCK-C to glucose metabolism and lipid metabolism in the process.