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High-Efficiency System for the Construction of Adenovirus Vectors and Its Application to the Generation of Representative Adenovirus-Based cDNA Expression Libraries

Moritz Hillgenberg,^1* Christian Hofmann,^1, Herbert Stadler,² and Peter Löser^1,

DeveloGen AG NL Berlin, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany,¹ Affectis Pharmaceuticals AG, Kraepelinstrasse 2, D-80804 Munich, Germany²

Received 31 January 2006/ Accepted 13 March 2006

We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10⁶ independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human ₁-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

Present address: Apit Laboratories GmbH, Hermanswerder 16, D-14473 Potsdam, Germany.

Present address: Robert-Koch-Institut, Seestr. 10, D-13353 Berlin, Germany.