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Updated: May 23, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the
Ming Liu1, Yi-sheng Wang, Yue-bai Li
1Department of Orthopedic Surgery, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
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.
Insights
Researchers developed recombinant adenovirus vectors to deliver small interfering RNA (siRNA) targeting peroxisome proliferator-activated receptor-γ (PPARγ). This approach aims to down-regulate PPARγ, potentially preventing steroid-induced osteonecrosis of the femoral head (ONFH).
Area of Science:
- Molecular biology
- Gene therapy
- Biotechnology
Background:
- Steroid-induced osteonecrosis of the femoral head (ONFH) is a debilitating condition with limited treatment options.
- Peroxisome proliferator-activated receptor-γ (PPARγ) is implicated as a key pathogenic gene in ONFH development.
- RNA interference (RNAi) offers a method to reduce specific gene expression.
Purpose of the Study:
- To construct recombinant adenovirus vectors for delivering small interfering RNA (siRNA) targeting PPARγ.
- To investigate the potential of down-regulating PPARγ expression as a strategy for preventing steroid-induced ONFH.
Main Methods:
- Synthesized three distinct siRNA sequences targeting the PPARγ gene.
- Annealed, purified, and ligated siRNA sequences into CMV shuttle vectors.
- Constructed recombinant adenovirus vectors carrying PPARγ siRNA and determined their high viral titers.
Main Results:
- Successfully constructed three recombinant shuttle vectors (1.0-CMV-971, 1.0-CMV-1253, 1.0-CMV-1367) containing PPARγ siRNA sequences.
- Confirmed the sequences of the recombinant vectors.
- Obtained high titers (>10^10 PFU/ml) of recombinant adenovirus carrying PPARγ siRNA.
Conclusions:
- Successfully developed recombinant adenovirus shuttle vectors for PPARγ siRNA delivery.
- Achieved high titers of recombinant adenovirus, indicating a viable tool for further research into ONFH prevention.
