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.
Adenoviridae ; Calcium ; Chromatography, High Pressure Liquid ; Dependovirus* ; DNA ; DNA, Viral ; Genetic Therapy* ; Genome ; Humans* ; Plasmids ; Product Packaging ; Transfection ; Uterine Cervical Neoplasms*