An Inducible Nitric Oxide Synthase-Luciferase Reporter System for In Vivo Testing of Anti-inflammatory Compounds in Transgenic Mice

Abstract

The inducible NO synthase gene (iNOS) plays a role in a number of chronic and acute conditions, including septic shock and contact hypersensitivity autoimmune diseases, such as rheumatoid arthritis, gastrointestinal disorders, and myocardial ischemia. The iNOS gene is primarily under transcriptional control and is induced in a variety of conditions. The ability to monitor and quantify iNOS expression in vivo may facilitate a better understanding of the role of iNOS in different diseases. In this study, we describe a transgenic mouse (iNos-luc) in which the luciferase reporter is under control of the murine iNOS promoter. In an acute sepsis model produced by injection of IFN-γ and LPS, we observed an induction of iNOS-driven luciferase activity in the mouse liver. This transgene induction is dose and time dependent and correlated with an increase of liver iNOS protein and iNOS mRNA levels. With this model, we tested 11 compounds previously shown to inhibit iNOS induction in vitro or in vivo. Administration of dexamethasone, epigallocatechin gallate, α-phenyl-N-tert-butyl nitrone, and ebselen significantly suppressed iNOS transgene induction by IFN-γ and LPS. We further evaluated the use of the iNos-luc transgenic mice in a zymosan-induced arthritis model. Intra-articular injection of zymosan induced iNos-luc expression in the knee joint. The establishment of the iNos-luc transgenic model provides a valuable tool for studying processes in which the iNOS gene is induced and for screening anti-inflammatory compounds in vivo.

In vivo bioluminescence for tracking cell fate and function

“…Thus the information gathered from BLI studies in small animals can be used to design solid clinical trials that will help improve gene therapy for cardiac repair. In cardiovascular research, BLI has been used to address a variety of questions that range from determining the effects of transgene expression (e.g., BCL2) on cardiomyoblast survival and cardiac repair (40) to testing the efficiency of anti-inflammatory drugs on the expression of specific genes (e.g., inducible nitric oxide synthase) (99). Additionally, BLI has been used to optimize vector systems (33) and treatment regimens (71) for delivery of therapeutic genes (e.g., hypoxia-inducible factor-1␣) to the heart.…”

Application of a disease-regulated promoter is a safer mode of local IL-4 gene therapy for arthritis

“…Minimizing the undesired effects of biologicals, in this case the potent proinflammatory and chemotactic properties of IL-4, requires promoters with low basal activity under naive conditions. Promoter regions from acute phase proteins (APP), [28][29][30][31][32][33] inducible nitric oxide synthase (iNOS), 34 proinflammatory cytokines, 13,14 and a nuclear factor-kappaB (NF-kB) responsive promoter 12 have been predominantly explored for their potential to effectuate arthritis-induced transgene expression. Although several of these appeared applicable for disease-regulated gene therapy, intrinsic properties limit their suitability for minimizing transgene expression under naive conditions.…”

In vivo imaging of tissue eosinophilia and eosinopoietic responses to schistosome worms and eggs

“…Our study highlights several areas worthy of further investigation and establishes the usefulness of EPX-luc reporter mice and in vivo bioluminescence imaging for studying the unique aspects of immune responses to helminth parasites. Luciferase trans-gene expression driven by other immunologically relevant promoters, such as the IL-4 promoter (Lee et al, 2003), the IL-6 promoter (Zhang et al, 2004), the IL-12 p40 promoter (Becker et al, 2003), the inducible nitric oxide synthase (iNOS) promoter (Zhang et al, 2003) and nuclear factor κ-B (NF-κB)-dependent promoters (Sadikot et al, 2003), combined with in vivo biophotonic imaging techniques, will likely produce more new data on the in vivo activity of immunologically important genes during immune responses to parasites. In vivo bioluminescence in the livers and intestines of Schistosoma mansoni-infected EPXluc mice.…”

“…In vivo imaging was performed using the IVIS ® Imaging System 100 Series (Xenogen Corp., Alameda, CA) (Becker et al, 2003;Lee et al, 2003;Sadikot et al, 2003;Zhang et al, 2003Zhang et al, , 2004. Prior to image collection, animals were anesthetised with isoflurane and the luciferase substrate luciferin was administered by intraperitoneal injection at a dose rate of 150 mg/kg body weight.…”