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Updated: Apr 16, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
Construction of recombinant adenoviral vector carrying cyclinA2 gene
Yu Han1, Liu Hong, Cuiping Zhong
1Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University.
Abstract:
Cell cycle related molecules in mammalian cochleae could provide a new avenue to restore hearing loss caused by a variety of genetic and environmental insults. CyclinA2 is one of the most important regulators of cell cycle, but its role in the mammalian cochlea is still unknown. So, it is necessary to construct an adenovirus vector carrying cyclinA2 gene for clarifying its function in the cochlea. In this study, the cyclinA2 genes were cloned into the shuttle plasmid pDC316-mCMV-EGFP to construct pDC316-CyclinA2-mCMV-EGFP, which was co-transfected with the rescue plasmid pBHGlox∆E1,3Cre into 293 cells to obtain the recombinant adenovirus Ad.CyclinA2-EGFP. Then, the plasmid pDC316-CyclinA2-mCMV-EGFP and recombinant adenovirus Ad.CyclinA2-EGFP were identified by restriction enzymes and reverse transcription-polymerase chain reaction (RT-PCR). The recombinant adenovirus vector was purified by CsCl banding, and was titrated. Finally, the recombinant adenovirus vector carrying cyclinA2 gene was constructed and confirmed by restriction enzyme analysis and RT-PCR. The titer of the recombinant adenovirus vectors reached 2.5 × 10(‒11) v.p/mL. Thus, we had successfully established the Ad.CyclinA2-EGFP vector, and it could express efficiently in various cells of cochlea. This study established the foundation for the further research of cyclinA2 gene's function in the cochlea.
Insights
Researchers developed a novel adenovirus vector (Ad.CyclinA2-EGFP) to study cyclinA2 gene function in the mammalian cochlea. This tool is crucial for understanding hearing loss mechanisms and developing future hearing restoration therapies.
Area of Science:
- Otolaryngology
- Molecular Biology
- Gene Therapy
Background:
- Hearing loss stems from diverse genetic and environmental factors.
- Cell cycle regulators, like cyclinA2, are potential targets for hearing restoration.
- The function of cyclinA2 in the mammalian cochlea remains largely unexplored.
Purpose of the Study:
- To construct and validate a recombinant adenovirus vector for expressing the cyclinA2 gene in the cochlea.
- To establish a foundation for investigating the role of cyclinA2 in cochlear biology and hearing loss.
Main Methods:
- Cloning of cyclinA2 genes into a shuttle plasmid (pDC316-mCMV-EGFP).
- Construction of recombinant adenovirus (Ad.CyclinA2-EGFP) via co-transfection with a rescue plasmid into 293 cells.
- Identification and confirmation using restriction enzyme analysis and reverse transcription-polymerase chain reaction (RT-PCR).
- Purification and titration of the adenovirus vector.
Main Results:
- Successful construction and confirmation of the Ad.CyclinA2-EGFP vector.
- Validated expression of cyclinA2 in cochlear cells.
- Achieved a high titer of 2.5 × 10(11) viral particles/mL for the vector.
Conclusions:
- The Ad.CyclinA2-EGFP vector was successfully established and validated.
- This vector provides an efficient tool for studying cyclinA2 gene function in the cochlea.
- The study lays the groundwork for future research into hearing restoration strategies targeting cyclinA2.
