Background: Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.
Methods: According to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.
Results: After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.
Conclusion: In this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.