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Korean Journal of Obstetrics & Gynecology 2004;47(4):695-701.
Published online April 1, 2004.
Construction of Recombinant Adeno-Associated Virus Vector (AAVCMVp53) for Human Cervical Cancer Gene Therapy.
Yong Wook Kim, Min Je Seo, Su Mi Bae, Jin Keoun Jun, Tae Hyung Kim, Tae Young Jin, Suk Woo Lee, Yong Wan Kim, Chong Kook Kim, Joon Mo Lee, Sung Eun Namkoong, Woong Shick Ahn
1Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea.
2Research Institute of Cancer, Catholic Research Institute of Medical Science, The Catholic University of Korea.
3College of Pharmacy, Seoul National University, Korea.
Abstract
OBJECTIVE
To eliminate the potential problem of adenovirus contamination during recombinant adeno-associated virus (rAAV) vector production, we investigated new rAAV production method by a triple transfection of vector plasmid, packaging plasmid, and adenovirus helper plasmid. METHODS: This study was carried by triple transfection for the production of recombinant adeno-associated virus vector. This new production system was conducted with a plasmid construct which contained a mini-adenovirus genome capable of propagation of rAAV in the presence of adeno-assoceated viral rep and cap genes. To examine the helper functions of adenoviral plasmid on the production of rAAV vector, we carried out cotransfection with three plasmids, AAV vector, packaging construct, and adenovirus helper plasmids. The optimized plasmid quantity of transfection by calcium phosphate precipitation method was 25 micro gram of total plasmid DNA per 10 cm diameter plate of 293 cell. RESULTS: We found that rAAV yields peaked at 48 hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/mL based on the quantification of viral DNA. CONCLUSION: We constructed recombinant AAVp53 without adenovirus contamination. We thought that we construct high titer rAAVp53 particle through HPLC column in the near future.
Key Words: Adeno-associated virus, Cotransfection, Packaging plasmid, Helper plasmid, Cervical cancer


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