Generation of a highly inducible Gal4 → Fluc universal reporter mouse for in vivo bioluminescence imaging

Abstract

Full understanding of the functional complexity of the protein interactome requires mapping of biomolecular complexes within the cellular environment over biologically relevant time scales. New approaches to imaging interacting protein partners in vivo will allow the study of functional proteomics of human biology and disease within the context of living animals. Herein, we describe a universal transgenic reporter mouse strain that expresses firefly luciferase (Fluc) under the regulatory control of a concatenated Gal4 promoter (Tg G4F(؉/؊) ). Using an adenovirus to deliver a fused binding-domainactivator chimera (Gal4BD-VP16), induction of bioluminescence in Tg G4F(؉/؊) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The Tg G4F(؉/؊) reporter strain allowed noninvasive detection of viral infectivity, duration of the infection as well as viral clearance in various tissues in vivo. To demonstrate protein-protein interactions in live mice, the well characterized interaction between tumor suppressor p53 (fused to Gal4BD) and large T antigen (TAg) (fused to VP16) was visualized in vivo by using a two-hybrid strategy. Hepatocytes of Tg G4F(؉/؊) mice transfected with p53/TAg demonstrated 48-fold greater induction of Fluc expression in vivo than noninteracting pairs. Furthermore, to demonstrate the feasibility of monitoring experimental therapy with siRNA in vivo, targeted knockdown of p53 resulted in markedly reduced light output, whereas use of a control siRNA had no effect on protein interaction-dependent induction of Fluc. Thus, this highly inducible Gal43Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.luciferase ͉ molecular imaging ͉ p53 ͉ protein-protein interactions ͉ transgenic reporter mouse

Bioluminescence imaging: progress and applications

“…2a). Using this system, Pichler et al have recently generated transgenic mice where Fluc is cloned under the control of the GAL4 promoter [19]. These mice were used to image a well known interaction between the tumor suppressor p53 (fused to GAL4) and large T antigen (TAg,fused to VP16) (Fig.…”

Monitoring Molecular-Specific Pharmacodynamics of Rapamycin In vivo with Inducible Gal4→Fluc Transgenic Reporter Mice

“…However, one of the major challenges in in vivo pharmacodynamic monitoring is the lack of high fidelity reporter models to visualize pharmacodynamics of therapeutic agents in a mechanistically appropriate manner. In this regard, using a universal Gal4 → Fluc reporter mouse (20), the inducible association of FRB and FKBP12 upon introduction of Rap enabled conditional activation of a luciferase reporter gene (Fig 1) (21–25). This platform could lead to broader applications of reporter models to study pharmacodynamics in the context of whole animals.…”

Imaging: Strategies, Controversies, and Opportunities

“…This approach provides molecule-specific pharmacodynamic information noninvasively in vivo, allowing a time course for response to treatment to determine the specific dosage for a desired molecular action (32,48,49). As genetically encoded imaging reporters are engineered into mouse lines (50,51), preclinical pharmacodynamic studies will be made more accurate due to direct observation of drug action on specific targets over time. These observations will confirm the mechanism of disease and drug action, validate that the drug is affecting the expected target, and validate the drug's efficacy (52).…”