Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration.
Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.
Zheng
Z
Pei
Saki
S
Kondo
Yumi
Y
Kanegae
Izumu
I
Saito
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005822:Genetic Vectors / Q000032:analysis* / Q000235:genetics; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014161:Transduction, Genetic
D004279:DNA, Viral