DEVELOPMENT AND CHARACTERIZATION OF A BINARY GENE-EXPRESSION SYSTEM BASED ON BACTERIOPHAGE-T7 COMPONENTS IN ADENOVIRUS VECTORS

To explore the utility of the bacteriophage T7 binary system in adenov irus (Ad) vectors we constructed three Ad5-based vectors containing th e T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase prod uced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-gala ctosidase (beta Gal) (lacZ) gene under the control of the T7 gene 10 p romoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) in ternal ribosome entry site (IRES) (AdBHG10T7 beta Gal). Coinfections w ere performed with the various AdT7pol vectors and the reporter vector , and expression was analysed in three different human cell lines: 293 , A549 and MRC-5. Depending on the AdT7pol vector used, different leve ls of expression were obtained from the reporter gene. In 293 cells, e xpression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7 beta Gal. In A549 and MRC-5 cells very l ittle expression was detected using AdT7pol1 or pol2 and efficient exp ression was only obtained when relatively high moi values of the repli cation-competent vector were used in the coinfections. We also constru cted a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evalu ate the 'leakiness' of the Ad-T7 system detected very little expressio n from the T7pro in the absence of T7 polymerase suggesting this syste m may be useful for the cloning and expression of genes encoding cytot oxic proteins. (C) 1997 Elsevier Science B.V.