Construction of recombinant adenoviral vector carrying cyclinA2 gene

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.