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J Virol, March 1998, p. 2289-2296, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassettedagger

Bernard Massie,1,* France Couture,1 Linda Lamoureux,1 Dick D. Mosser,1 Claire Guilbault,2 Pierre Jolicoeur,1 François Bélanger,1 and Yves Langelier2

Institut de Recherches en Biotechnologie, Montréal, Québec, Canada H4P 2R2,1 and Centre de Recherche Louis Charles Simard, Institut du Cancer de Montréal, Montréal, Québec, Canada H2L 4M12

Received 30 May 1997/Accepted 21 November 1997

We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Delta 2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce Delta R1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5Delta R1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montréal, Québec, Canada H4P 2R2. Phone: (514) 496-6131. Fax: (514) 496-5143. E-mail: Bernard.Massie{at}NRC.ca.

dagger NRC publication no. 39989.




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