Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase.
Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.
Schaack
J
J
Department of Microbiology, University of Colorado Cancer Center, University of Colorado Health Sciences, Denver 80262, USA.
Langer
S
S
Guo
X
X
D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004279:DNA, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D004279:DNA, Viral; D001616:beta-Galactosidase