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Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction
- Source :
- Gene Therapy. 11:1599-1605
- Publication Year :
- 2004
- Publisher :
- Springer Science and Business Media LLC, 2004.
-
Abstract
- We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.
- Subjects :
- Genetic Vectors
Genome, Viral
Gene delivery
Biology
medicine.disease_cause
Genome
Adenoviridae
law.invention
Plasmid
Luciferases, Firefly
law
Escherichia coli
Genetics
medicine
Animals
Coding region
Transgenes
Cloning, Molecular
Molecular Biology
Gene
Recombination, Genetic
Expression vector
Recombinant DNA
Molecular Medicine
Genetic Engineering
Plasmids
Subjects
Details
- ISSN :
- 14765462 and 09697128
- Volume :
- 11
- Database :
- OpenAIRE
- Journal :
- Gene Therapy
- Accession number :
- edsair.doi.dedup.....2c543c0894ce71e54d9d1a131e192300