Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor.
When recombinant plasmids containing the entire adeno-associated virus (AAV) genome are transfected into permissive cells infected with a helper adenovirus, infectious AAV particles are efficiently generated. These plasmids can be used to generate mutant AAV genomes or recombinant AAV vectors. Packaging of mutant AAV genomes has required complementation with a second AAV plasmid in the transfection assay which may lead to generation of significant amounts of wild-type AAV recombinants. One approach to alleviate this problem was to generate conditional lethal mutants. We constructed an AAV plasmid recombinant having a nonsense mutation in the AAV rep gene by using oligonucleotide-directed mutagenesis to convert a serine codon to an amber codon. We show that this mutant AAV can be grown on monkey cell lines containing an inducible human serine tRNA amber suppressor. The amber suppression is quite efficient and yields a burst of mutant AAV particles at about 10% of the titer of wild-type AAV. The reversion frequency of the amber mutation appears to be less than 10(-5).
Chejanovsky
N
N
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Carter
B J
BJ
D000818:Animals; D002460:Cell Line; D004261:DNA Replication; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development*; D005786:Gene Expression Regulation; D000882:Haplorhini; D009154:Mutation; D010957:Plasmids; D012343:RNA, Transfer / Q000235:genetics; D013489:Suppression, Genetic; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
D014764:Viral Proteins; D012343:RNA, Transfer