Packaging and Functional Identification of Recombinant A deno-associated Virus Encoding cdc2-siRNA
- Author:
WEI JIAJUN
1
;
ZHANG MIN
;
BU BITAO
;
ZHANG SUMING
;
XU JINZHI
Author Information
1. Department of Neurology, Tongfi Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
- Keywords:
small interfering RNA;
recombinant adeno-associated virus;
gene therapy
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2008;28(6):626-629
- CountryChina
- Language:Chinese
-
Abstract:
Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegenerative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-odc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful, rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.
