A simple method for the rapid generation of recombinant adenovirus vectors.
Gene Ther
; 7(12): 1034-8, 2000 Jun.
Article
in En
| MEDLINE
| ID: mdl-10871752
ABSTRACT
Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenoviridae
/
Genetic Vectors
Limits:
Humans
Language:
En
Journal:
Gene Ther
Journal subject:
GENETICA MEDICA
/
TERAPEUTICA
Year:
2000
Document type:
Article
Affiliation country:
Estados Unidos