Imaging Luciferase-Expressing Viruses
Optical imaging of luciferage gene expression has become a powerful tool to track cells and viruses in vivo in small animal models. Luciferase imaging has been used to study the location of infection by replication-defective and replication-competent viru
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1. Introduction The technologies available to the basic scientist to track and localize viruses and tumor cells have historically been quite primitive. In most cases, virus and cell trafficking has been assessed by the use of terminal assays in which the animal must be sacrificed and the cells or viruses are tracked after the animal is “taken apart” either at the organ level or in tissue sections. These “grind and find” assays are quite laborious requiring that one actually sections the whole animal to be certain of the tissue localization of the virus to ensure that all sites are observed and unexpected localization sites are not missed. Furthermore, these terminal assays obviate the ability to perform kinetic studies in one animal over many time points. Given these difficulties, noninvasive and nonterminal virus and cancer cell tracking was needed. One approach that partially satisfies
David H. Kirn et al. (eds.), Oncolytic Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 797, DOI 10.1007/978-1-61779-340-0_6, © Springer Science+Business Media, LLC 2012
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this need is to “arm” viruses or cancer cells with reporter genes that can be detected by imaging. Reporter genes encode proteins that are easily detected in cells and in intact animals with sensitive imaging systems. The most used reporter genes include betagalactosidase, luciferases, green fluorescent protein (GFP) and its varied color derivatives, and alkaline phosphatase. Of these, luciferase and fluorescent proteins can be used to varying degrees for optical imaging at visible wavelengths of light in small animals. One can also use reporter genes, such as thymidine kinase or the sodium iodide symporter, for high-energy PET and SPECT imaging in small animals, large animals, and in humans. For most researchers, optical imaging is simpler and more easily obtainable in the laboratory setting than radioactive imaging for PET or SPECT. Since PET and SPECT imaging are the subject of another chapter, they are not discussed further here. One can in some cases directly image reporters, like GFP and other fluorescent proteins in living animals. In practice, high background fluorescence and scatter in the green, red, and far red wavelengths make the “noise” of imaging too high to easily detect most current fluorescent proteins in vivo (1, 2). Newer, far red fluorescent proteins to date are still difficult to image in mice (M.A. Barry et al., unpublished observations), but future near-infrared fluorophores may circumvent this difficulty. Given these issues, luciferase imaging is arguably the best choice for noninvasive, inexpensive, and nonradioactive imaging in small animals. Given this, we have “armed” replication-defective and replication-competent adenovirus serotype 5 (Ad5) viruses with luciferase and GFP–luciferase reporter genes to track (1) sites of infection, (2) persistence of infection, (3) spread of virus, and (4) elimination of virus due to immune responses (1, 3–11). In addition, one can monitor immune responses against these p
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