Package of recombinant adeno-associated virus encoding cell division cycle 2-siRNA

Jiajun WEI; Zhuoran WANG

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.