Effects of PPARγ Overexpression by Recombinant Adenovirus on Fat Deposition in Cattle (Bos taurus) Muscle Cells
HUA Liu-Shuai1, 2, WANG Jing2, XU Zhao-Xue2, WANG Er-Yao2, *, CHEN Hong1, *
1 College of Animal Science and Technology, Northwest A&F University/Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling 712100 China;; 2 Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation/Institute of Animal Husbandry and Veterinary Science, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Abstract：Intramuscular fat deposition is a complex biological process involving muscle cell differentiation, adipocyte differentiation and triglyceride metabolism. Peroxisome proliferator-activated receptor γ (PPARγ) gene plays a central role in the differentiation of adipocytes in animals, but the relationship between PPARγ gene expression and intramuscular fat content still not clear. The aim of this study was to package the cattle (Bos taurus) PPARγ Adenovirus and analyze the effects of PPARγ overexpression on fat deposition in muscle cells. After subcloning PPARγ into the vector pAdTrack-CMV, a shuttle vector pAdTrack-CMV-PPARγ was constructed. The linearized shuttle vector pAdTrack-CMV-PPARγ and the adenoviral backbone vector pAdEasy-1 were co-transformed into the recombinant bacterial BJ5183 (Escherichia coli), and after homologous recombination, the adenoviral backbone vector pAdEasy-1-PPARγ containing the gene of interest was constructed. The linearized pAdEasy-1-PPARγ was transferred into 293A cells (Homo sapiens) to package and amplify the PPARγ Adenovirus. After infection of bovine muscle cells with PPARγ Adenovirus, the expression changes of the fat deposition related genes were detected by quantitative real-time PCR (qRT-PCR). The results showed that the PPARγ Adenovirus vector was successfully constructed. After the package and 2 times of amplification, the PPARγ Adenovirus with a titer of 2×1011 plaque forming unit (PFU) /mL were obtained. Infection of bovine muscle cells with the PPARγ Adenovirus yielded an infection efficiency of more than 90%. After overexpression of PPARγ in bovine muscle cells, the positive regulation genes of fat deposition, including fat acid binding proteins 4 (FABP4), glucose transporter 4 (Glut4) and adipose-specific phospholipase A2 (AdPLA2) were up-regulated to 1.89, 1.51 and 1.56 times (P≤0.05). While the negative regulation genes of fat deposition including GATA binding protein 2 (GATA2), hormone-sensitive lipase (HSL) and nuclear receptor subfamily 2, group F, member 2 (Nf2f2) were down-regulated to 0.45, 0.57 and 0.25 times (P≤0.05). The expression level of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1a) was up-regulated to 141.41 times (P≤0.01), and the expression of myogenic factor 5 (Myf5) gene was not changed much. This study successfully packaged PPARγ Adenovirus, providing a tool for further study of the function of this gene, and proved that overexpression of PPARγ in bovine muscle cells has a tendency to promote fat deposition in muscle cells.
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