Package of recombinant adeno-associated virus encoding cell division cycle 2-siRNA
10.3969/j.issn.1673-8225.2009.28.031
- VernacularTitle:携带cdc2-siRNA重组腺相关病毒的包装
- Author:
Jiajun WEI
;
Zhuoran WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(28):5549-5553
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Cell division cycle 2 (cdc2) plays an important role in the course of neuronal degeneration in neurodegenerative diseases such as Alzheimer's disease. The silencing of cdc2 gene with adeno-associated virus vector might protect the neurons in neurodegenerative diseases.OBJECTIVE: To pack recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. DESIGN, TIME AND SETTING: Blank control experiment was performed in the laboratory of Department of Neurology at Wuhan Tongji Hospital between October 2007 and August 2008. MATERIALS: Helper Free adeno-associated virus system (pAAV-MCS-EGFP, p-RC, p-Helper) and AAV-293 cells were purchased from Stratagene. METHODS: The pAAV-MCS-U6-cdc2-siRNA-EGFP expressing plasmid was constructed by molecular biological techniques. The cotransfection of this plasmid, together with p-RC and p-Helper into AAV-293 calls was mediated by calcium acid phosphate. The rAAV encoding cdc2-siRNA (rAAV-U6-cdc2-siRNA-EGFP) was packed and harvested. The silencing effect of this virus on cdc2 gene expression in AAV-293 cells was assessed by Western Blot, and its titer was determined by spot hybridization method. MAIN OUTCOME MEASURES: sequencing of pAAV-MCS-U6-cdc2-siRNA-EGFP plasmid inserting U6-cdc2-siRNA; 3 plasmid pAAV-MCS-U6-cdc2-siRNA-EGFP, p-RC, p-Helper cotransfecting AAV-293 cells; cdc2 expression after rAAV-U6-cdc2-siRNA-EGFP infected AAV-293 cells.RESULTS: ①The successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP plasmid was proved by DNA sequencing. ②Green fluorescent protein was expressed in AAV-293 cells, and the contransfection was working well. ③The cdc2 gene expression in AAV-293 cells was down-regulated markedly after infection of rAAV-U6-cdc2-siRNA-EGFP. ④The titer of rAAV-U6-cdc2-siRNA-EGFP was 1×10<'12> v.g/mL.CONCLUSION: The package of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively.
