Generation of recombinant adenovirus vector with infectious adenoviral genome released from cosmid-based vector by simple procedure allowing complex manipulation.
To perform the complex manipulation of the adenoviral genome for the construction of recombinant adenovirus vectors, we developed a cosmid vector (pacad1A) from which an infectious E1 and E3-deleted adenoviral genome can be released with PacI digestion. The cosmid vector, pacad1A, has unique restriction enzyme sites that are created for the insertion of foreign genes into the deleted E1 or E3 region of the adenoviral genome. To demonstrate the feasibility of the construction of adenovirus vectors with our developed vector, we showed that a recombinant adenovirus bearing a self-contained tetracycline-regulated expression system could be generated by transfection of cells with an infectious adenoviral genome that was released from pacad1A-derived plasmid DNA. The recombinant adenovirus vector was obtained easily by this method, and the expression of a transgene was proved to be regulated with tetracycline in CHO-K1 cells.
Kojima
H
H
Gifu International Institute of Biotechnology, Japan.
Ohishi
N
N
Yagi
K
K
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D017498:Adenovirus E1 Proteins / Q000235:genetics; D017503:Adenovirus E3 Proteins / Q000235:genetics; D003360:Cosmids / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection / Q000379:methods*; D019076:Transgenes
D017498:Adenovirus E1 Proteins; D017503:Adenovirus E3 Proteins; D008156:Luciferases; C046184:endodeoxyribonuclease PaeI; D015252:Deoxyribonucleases, Type II Site-Specific; C068375:endodeoxyribonuclease PacI; D013752:Tetracycline