Construction of an adeno-associated virus vector pAGX(+)
Objective To construct an adeno associated virus vector for gene delivery to and gene expression in mammalian cells. Methods Gene recombinant technique for the construction of the vector, Southern blot, slot blot and laser confocal microscopy for determining the efficiency of EYFP gene rescue, gene transfer,expression and integration into host genome mediated by pAGX(+). Results An adeno associated virus vector named pAGX(+) was successfully constructed. The infection titer and the replication titer of rAAV/EYFP virion was 1.39×10 7 TU/ml and 1.94×10 11 particles/ml, respectively. Southern blot proved that the EYFP gene was successfully rescued. EYFP gene was successfully transducted to and expressed in mammalian cell line COS 7 cells. Southern analysis showed that the EYFP gene was integrated into the genomic DNA of COS 7 cells mediated by the transduction of rAAV/EYFP. On the contrary, the plasmid pEYFP CMV mediated EYFP gene transfer did not show EYFP gene integration. Conclusion An adeno associated virus vector pAGX(+) was successfully constructed, and the transfer, expression and integration of a gene were successfully mediated in mammalian cells by the rAAV.