Adenovirus vector library: an approach to the discovery of gene and protein function.
A method was developed to generate a complex cDNA expression library within an adenovirus type 5 (Ad5)-based vector backbone, termed AdLibrary. Construction of the AdLibrary entailed the conversion of an Ad5 genome-containing cosmid to infectious virus particles. The Ad5 genome was modified by replacing the E1A and E1B genes with a Rous sarcoma virus-driven expression cassette. Conversion was accomplished by liberating the viral genome by restriction enzyme digestion and transfection in HEK 293 cells, which support the growth of E1A/E1B-deficient virus. A test AdLibrary demonstrated the possibility of converting and identifying a marker gene present at a frequency of 1/10(5) in the cosmid library. To demonstrate the utility of this technology, an AdLibrary was used to isolate a viral gene by its biological function. Virus growth was selected for with an AdLibrary on A549 cells, which do not complement for E1A/E1B function. The AdLibrary was generated with cDNAs derived from HeLa cells productively infected with Ad5. A cDNA corresponding to Ad5 E1A 13S was selected and isolated from the AdLibrary using this strategy. Since multiple genes are assayed simultaneously, this technology should expedite the discovery of genes affecting defined biological activities. This AdLibrary approach provides an opportunity to exploit the efficient gene delivery capabilities of adenovirus vectors for the rapid discovery of gene and protein function.
McVey
Duncan
D
GenVec Inc., 65 West Watkins Mill Road, Gaithersburg, MD 20878, USA.
Zuber
Mohammed
M
GenVec Inc., 65 West Watkins Mill Road, Gaithersburg, MD 20878, USA.
Brough
Douglas E
DE
GenVec Inc., 65 West Watkins Mill Road, Gaithersburg, MD 20878, USA.
Kovesdi
Imre
I
KILA Consultants, LLC, 7713 Warbler Lane, Rockville, MD 20855-1033, USA.
D000256:Adenoviridae / Q000737:chemistry / Q000235:genetics*; D017498:Adenovirus E1 Proteins / Q000502:physiology; D002460:Cell Line; D018076:DNA, Complementary; D004279:DNA, Viral; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans
D017498:Adenovirus E1 Proteins; D018076:DNA, Complementary; D004279:DNA, Viral