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

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 (TgG4F(+/-)). Using an adenovirus to deliver a fused binding-domainactivator chimera (Gal4BD-VP16), induction of bioluminescence in TgG4F(+/-) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The TgG4F(+/-) 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 TgG4F(+/-) 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 Gal4→Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.
Generation and analysis of the reporter transgene. (A) Schematic representation of the transgene vector that was injected into pronuclei of FVB oocytes. (B) HeLa Fluc #43, a cell line stably expressing the Gal4→Fluc reporter construct, was transfected with a positive control vector pM3-VP16 that expresses a fusion of Gal4BD to VP16 (right well). A 43-fold induction was observed when compared with mock-transfected cells (left well). (C) PCR genotyping from a negative mouse (WT), a positive littermate (TgG4F(+/-)), and pGL3 as positive control are shown. (D) Mouse embryonic fibroblasts (MEF) of a negative embryo (WT) and a positive embryo (TgG4F(+/-)) were treated with either vehicle or 4x107 pfu of Ad5Gal4BD-VP16 (Ad-Gal4). Cells were imaged 24 h after infection. MEFs from the positive embryo were induced 3-log fold when compared with the uninducible negative MEFs.
Reference:	Pichler A, Prior JL, Luker GD, Piwnica-Worms D. Generation of a highly-inducible Gal4→Fluc universal reporter mouse for in vivo bioluminescence imaging. Proc Natl Acad Sci USA 2008; 105(41): 15932-7.	PubMed Link

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