Preparation of recombinant adenoviruses of PPARγ and its transcriptional coactivator MED1 and their biological function analysis

Objective: To obtain a large number of recombinant adenoviruses of PPARγ (Ad/PPARγ) and MED1 (Ad/MED1) and investigate the biological function of nuclear receptor PPARγ and its coactivator mediator 1(MED1). Methods: HEK293a cell line was amplified and used to package the adenovirus stocks (Ad/PPARγ or Ad/MED1). CsCl gradient centrifuge was used to purify the adenovirus, and virus particles were determined by A₂₆₀. After cell infection or mouse injection with adenovirus, the biological function of Ad/PPARγ and Ad/MED1 was analyzed using HE staining, Real-time PCR or immunohistochemistry. Results: We obtained control Ad/LacZ, Ad/PPARγ and Ad/MED1, whose concentrations were 2.51×10¹² VP/mL, 1.57×10¹² VP/mL and 2.59×10¹² VP/mL, respectively. Ad/PPARγ injected into the tail vein led to hepatic steatosis in C57BL/6J mice. HE and oil red O staining results showed that many lipid droplets accumulated in hepatocytes. Real time PCR and immunohistochemistry showed that the expressions of PPARγ mRNA and protein were remarkably increased. Also, MED1 mRNA expression was significantly increased in 3T3-L1 cell line or C57BL/6J mice after Ad/MED1 treatment compared with that in control group. Conclusion: Ad/PPARγ and Ad/MED1 were prepared successfully, which provides materials for cell infection, especially for gene function and network regulation in vivo.