Packaging and functional identification of recombinant adeno-associated virus encoding cdc2-siRNA.
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-cdc2-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.
Wei
Jiajun
J
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. nicer6wei@yahoo.com.cn
Zhang
Min
M
Bu
Bitao
B
Zhang
Suming
S
Xu
Jinzhi
J
D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000235:genetics*; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics*; D011995:Recombination, Genetic
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase