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Packaging and functional identification of recombinant adeno-associated virus encoding cdc2-siRNA.

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-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.
AuthorsJiajun Wei, Min Zhang, Bitao Bu, Suming Zhang, Jinzhi Xu
JournalJournal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban (J Huazhong Univ Sci Technolog Med Sci) Vol. 28 Issue 6 Pg. 626-9 (Dec 2008) ISSN: 1672-0733 [Print] China
PMID19107353 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • RNA, Small Interfering
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • CDC2 Protein Kinase
Topics
  • Base Sequence
  • CDC2 Protein Kinase (genetics)
  • Cell Line
  • Dependovirus (genetics, metabolism)
  • Genetic Vectors
  • Green Fluorescent Proteins (genetics)
  • Humans
  • Molecular Sequence Data
  • RNA, Small Interfering (genetics)
  • Recombination, Genetic

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