Reporter genes for monitoring gene expression in mammalian cells

Publisher Summary The advent of reporter gene technology has greatly facilitated the knowledge of the mechanisms of many cellular processes Reporter genes encode proteins with phenotypic properties that are both distinct (from the system being studied) and conveniently monitored. Linkage of the cellular activity being assayed to the phenotypic expression of the reporter is accomplished by the fusion of appropriate DNA sequences to the reporter gene. These sequences are either regulatory in nature, typically being responsive to the cellular event under examination, or structural, encoding proteins that mediate such an event. After introduction of the chimera into cells, the qualitative or quantitative activity of the cellular event is extrapolated from the expression of the linked reporter gene product. Application of reporter–gene technology to mammalian cells was introduced in 1982 with the development of plasmid vectors, encoding the bacterial enzymes chloramphenicol acetyltransferase (CAT) or β-galactosidase (β-gal), to study eukaryotic gene regulation. As in these initial studies, reporter genes have been traditionally used to characterize and dissect transcriptionally active regulatory regions. Although characterization of cis-elements remains a common usage, reporter gene technology has attained increasing levels of sophistication and is now used in numerous other applications: characterization of transcription factors and associated proteins such as co-activators, delineation of signal transduction pathways, identification of protein–protein interactions, determination of cell fates, visualization of cellular trafficking, high-throughput screening of chemicals for adverse or therapeutic effects, and optimization and monitoring of DNA delivery systems. Numerous genes, both of prokaryotic and eukaryotic origin, have been proposed for use as reporter genes in mammalian cells. Only a handful of these, however, are widely used in this capacity. Aside from the obvious characteristic that reporter gene products cannot be toxic, the commonly used reporters share two other features: (1) the gene products exhibit phenotypic characteristics that are unique (i.e., foreign to mammalian cells) or can be easily distinguished from any similar endogenous activity, and (2) methods for detection of the reporter are sensitive, generally quantitative, and exhibit a broad linear dynamic range; they are easy to perform, reproducible and reasonably cost-effective. In terms of the reporter genes themselves, the single most significant development since that time is the introduction and evolution of green fluorescent protein (GFP) as a powerful and multi-faceted reporter protein. The chapter discusses this system as well as some of the other reporter gene systems.