Construction of adenoviral vectors |
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Authors: | Alan R Davis Nelson A Wivel Joseph L Palladino Luan Tao James M Wilson |
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Institution: | (1) Institute for Human Gene Therapy, University of Pennsylvania, 204 Wistar Institute, 3601 Spruce Street, 19104 Philadelphia, PA |
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Abstract: | Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors
requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves
deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after contransfection of
293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains
a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high
background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from
uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an
E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques
(green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages
when production lots of the recombinant vector are being made. |
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Keywords: | Recombinant adenovirus cotransfection shuttle vector transgene restriction fragment DNA negative selection green fluorescent protein |
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