Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase.
Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.
Hartigan-O'Connor
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D
Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA.
Amalfitano
A
A
Chamberlain
J S
JS
D000256:Adenoviridae / Q000235:genetics / Q000254:growth & development*; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D018741:Replication Origin; D014162:Transfection; D014764:Viral Proteins
D010750:Phosphoproteins; D011498:Protein Precursors; D014764:Viral Proteins; C085476:pTP protein, adenovirus; D004259:DNA-Directed DNA Polymerase