Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences
| Autor: | Edward Otto, Elio F. Vanin, Agnes Jones-Trower, W. French Anderson, Kathy Stambaugh, Gerard J. McGarrity, Stephen N. Mueller |
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| Rok vydání: | 1994 |
| Předmět: |
Sequence analysis
Genetic Vectors Molecular Sequence Data Moloney murine sarcoma virus Sequence alignment Biology Virus Replication Polymerase Chain Reaction law.invention Cell Line Retrovirus Proviruses Viral Envelope Proteins law Genetics Animals Molecular Biology Recombination Genetic Base Sequence Gene Transfer Techniques Chromosome Mapping Sequence Analysis DNA biology.organism_classification Virology Long terminal repeat DNA Viral Recombinant DNA Molecular Medicine Replication Competent Retrovirus Primer (molecular biology) Moloney murine leukemia virus Safety Homologous recombination Sequence Alignment |
| Zdroj: | Human gene therapy. 5(5) |
| ISSN: | 1043-0342 |
| Popis: | A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system. |
| Databáze: | OpenAIRE |
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