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Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor
Year:
1989
Source of publication :
Virology
Authors :
Chejanovsky, Nor
;
.
Volume :
171
Co-Authors:
Chejanovsky, N., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 304, Bethesda, MD 20892, United States
Carter, B.J., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 304, Bethesda, MD 20892, United States
Facilitators :
From page:
239
To page:
247
(
Total pages:
9
)
Abstract:
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. © 1989.
Note:
Related Files :
Animal
gene expression regulation
genetic engineering
mutation
parvovirus
recombinant plasmid
virus replication
Show More
Related Content
More details
DOI :
10.1016/0042-6822(89)90531-X
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
30043
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:51
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Scientific Publication
Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor
171
Chejanovsky, N., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 304, Bethesda, MD 20892, United States
Carter, B.J., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 304, Bethesda, MD 20892, United States
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. © 1989.
Scientific Publication
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