title: Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression.
abstract: Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.
authors.affiliation: Crump Institute for Molecular Imaging, and Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095, USA.
authors.forename: Sunetra
authors.initials: S
authors.lastname: Ray
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Paulmurugan
authors.forename: Isabel
authors.initials: I
authors.lastname: Hildebrandt
authors.forename: Meera
authors.initials: M
authors.lastname: Iyer
authors.forename: Lily
authors.initials: L
authors.lastname: Wu
authors.forename: Michael
authors.initials: M
authors.lastname: Carey
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000818:Animals; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D008297:Male; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: C070633:Gal-VP16; D015534:Trans-Activators; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly