A rapid and quantitative assay for measuring neighboring gene activation by vector proviruses.
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.
Hendrie
Paul C
PC
Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington 98195-7720, USA.
Huo
Yunwen
Y
Stolitenko
Raisa B
RB
Russell
David W
DW
D015152:Blotting, Northern; D002460:Cell Line; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006678:HIV / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D009052:Leukemia Virus, Murine / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053150:Simian foamy virus / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases