1. ¹Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington, USA
2. ²Department of Biochemistry, University of Washington, Seattle, Washington, USA

Correspondence: David W. Russell, Department of Medicine, University of Washington, Mailstop 357720, 1705 NE Pacific Street, Seattle, Washington 98195-7720, USA. E-mail: drussell@u.washington.edu

Received 5 March 2007; Accepted 29 November 2007; Published online 22 January 2008.

Abstract

A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.