Cloning And Expression Of PPARγ Gene Of Xuhuai Goat And Preparation Of Its Transgenic Sheep

Peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear transcription factors. Studies have shown that PPARs are consisted of three subtypes, namely PPARα,β,γ. PPARy’s biological effects in animal fat metabolism regulation mainly are to promote the early adipocyte differentiation and positive lipid metabolism. Its regulation to some gene expressions in lipid metabolism would ultimately lead to increased clearance of triglyceride, stimulation cell uptake of fatty acids, triglycerides and very low density lipoprotein. It also can increase high-density protein. So it is the physiological sensor of lipid balance, and it plays a pivotal role in maintaining dynamic balance of lipid metabolism. Currently, researches about this gene mainly focused on the lipid metabolism diseases of human and mouse, while for cattle, sheep and other livestocks mainly focused on meat quality issues. The study cloned Xuhuai goat PPARy gene, studied its expression in vitro and subcellular localization, and verified its biological function at the cellular level. At last, PPARy gene was mediated through testis injection to produce transgenic sheep. The main research contents were as follows:1. PPARy gene cloning, sequence analysis and construction of its recombinant eukaryotic expression vector. PPARy gene-specific primers were designed and synthesized. Xuhuai goat PPARy cDNA in adipose tissue was first cloned by RT-PCR method. The PPARy gene was cloned into pMD-19T vector. The digestion and sequencing results confirmed that the pMD19-T-PPAR was constructed correctly. Then the PPARy gene was sub cloned into the eukaryotic expression vector pEGFP-C1to construct the recombinant fusion expression vector named pEGFP-PPARy. Digestion and sequencing results confirmed that the pEGFP-PPARγ was constructed correctly. Sequence analysis revealed that PPARγ of Xuhuai goat had a homology of81%,89%,92%,98%,99%with chicken, mouse, pig, cattle and sheep, respectively. And PPARy animo acids sequence had a homology of92.9%,97.3%,98.3%,99.6%,99.8%with chicken, mouse, pig, cattle and sheep, respectively. PPARγ in different species was conservative.2. Studies of PPARy gene recombinant expression in vitro and subcellular localization. NIH-3T3cells were transfected pEGFP-PPARγ through polyethylene imine (PEI) and then observed under inverted microscope to identify recombinant proteins localization in NIH-3T3cells. Then software prediction, mRNA and total proteins were detected to identify the expression in NIH-3T3cells by RT-PCR and Western Blotting methods respectively. Software prediction showed that PPARy mainly expressed in cytoplasm which was consistent with the result under the inverted microscope. And the study achieved the PPARγ fusion expression at the cell level, and EGFP-PPARy protein was about80kDa. All these above provided basis for the construction of PPARy stable expression cell lines and the preparation of transgenic sheep.3. Differentiation of NIH-3T3fibroblasts into adipocytes induced by PPARy expression. Liposome-mediated pEGFP-PPARγ transfected NIH-3T3cell lines, then positive monoclonal cell lines were obtained by G418selection for15～20days. Positive monoclonal cell lines were added activators including5ug/ml insulin, luM dexamethasone, and0.5uM rosiglitazone to differentiate for15days. They were identified by oil red O staining and the expression of adipocyte specific genes with RT-PCR method. Results showed that about5days of induction, lipid droplet appeared around nuclear in some cells. About8-15days later, cells with lipid droplet increased to about30%. And adipocytes specific gene named Lipoprotein lipase gene (LPL) could be detected by RT-PCR which was about1500bp. An adipocyte differentiation model in vitro was successfully established. The work provided basis for further research on the molecular mechanism of adipocyte differentiation induced by PPARy.4. Preparation and detection of PPARy transgenic sheep. Through liposome transfection, the linearized pEGFP-PPARy was injected into mature Hu sheep’testicles. Repeated injection was conducted after one week from first injection. Forty days later, they mated with normal ewes to produce F1offspring. Conventional PCR, genomic DNA dot blotting and western blotting were used to detect pEGFP-PPARy in F1. Results indicated that PCR positive rate was20.6%, while the genome DNA dot blot positive rate was13.7%. And the positive rate of total protein western blot was13.7%. All these above initially demonstrated that pEGFP-PPARy could be integrated into the genome of Hu sheep and be transmitted to the next generation.