بررسی ترانسفکت وکتورهای نوترکیب آدنوویروسی فاقد ژن گزارشگر و تیتراسیون آن با روش‌های مولکولی: راهنمای عملی

Abstractbackground and objectivesbiologival features of adenoviruses have made them vectors of choice for gene transfer in basic research as well as in clinical treatment and vaccination. the key steps in the trasnsfer of adenovirus vector gene are transfection monitoring and viral titer determination. we investigated the transfection of non-reporter adenoviruses as well as viral titration using molecular methods.materials and methodsin this experimental study, after the recombinant adenovirus vector construction, the presence of transgenes in gene construct was evaluated by pcr with transgene specific primers and sequencing. after calcium-phosphate transfection of the packaging cell line (hek 293a), transfection was monitored by pcr using transgene specific primers (diagnostic and compound primers) to analyze dna extracted from the transfected packaging cell line. the viral titer was determined by real time pcr (rt-pcr) using diagnostic primer to analyze dna extracted from viral lysates.resultsthe presence of transgene in gene construct was confirmed by transgene specific primers and sequencing. evaluation of dna extracted from transfected packaging cells confirmed the efficiency of monitoring of transfection. the viral titer was estimated to be about 4 × 107 viral particles/ml by rt-pcr. conclusionscareful primer design is the key to the efficient monitoring of non-reporter adenovirus vector transfection. the appropriate genomic purification and the use of rt-pcr technique would also avoid the disadvantages of functional titration of adenoviruses.