To gain insightful information about the mechanisms through which genes are activated and repressed requires gene reporter systems that are sensitive, robust, and cost-effective. Although numerous reporter gene technologies are commercially available, none are as sophisticated and user-friendly as β-lactamase (BLA) when it comes to studying gene expression in live cells. This article presents an overview of the BLA technology and describes how it can be exploited for studying rare events such as homologous recombination in somatic cells and be used to deliver any DNA sequence of choice anywhere within the genome.

Introduction

Over the past several years, the combination of functional genomics, proteomics, and bioinformatics has contributed significantly to the identification of novel molecules that now serve as valid targets for therapeutic intervention against various diseases. Rapid generation of lead compounds that will inhibit or, in some cases, potentiate the activities of these target molecules necessitates the availability of robust, sensitive, and cost-effective assays. Previously, biochemical assays were used more than cell-based assays, but, due to emerging technologies, this trend is changing, and the demand for cell-based assays is gradually increasing. Although biochemical assays are well-defined, sensitive, and easy to miniaturize, they are nonphysiological and require purified protein. Moreover, biochemical assays can be challenging to develop for difficult-to-purify (but nonetheless therapeutically important) proteins, such as membrane receptors, ion channels, and G protein-coupled receptors (GPCRs). In contrast, cell-based assays offer investigators the luxury of probing biological activity of a protein or a pathway in its natural physiological milieu, and therefore are closer to recapitulating the actual in vivo situation.

Current Reporter Gene Detection Systems

The success of cell-based assays depends entirely on the quality of the reporter gene. To date, numerous reporter genes have been used for monitoring gene expression (1). While useful, most commercially available reporter technologies are not versatile enough to cover every aspect of cell-based assay development and screening (see Table 1). For instance, although luciferase-based assays provide acceptable sensitivity and an excellent dynamic range, they cannot be used for fluorescence-activated cell sorting (FACS), because the bioluminescent emission produced after the breakdown of D-luciferin is only transient. Thus, deriving stable cell lines from single cells expressing luciferase necessitates labor-intensive serial dilutions and makes this system ill-suited for cell-based assay development (1). Secreted alkaline phosphatase (seAP) is another reporter system in which the expressed seAP is detected when it activates a dioxetane derivative, which decomposes and emits light during the decomposition process. Like luciferase, the seAP system is incompatible with FACS, and developing seAP expressing cell lines from single cells also requires time-consuming and labor-intensive serial dilutions. The oldest reporter system, chloramphenicol acetyl transferase (CAT), requires a radioactive substrate; this feature reduces its utility for many researchers.

Table 1. Characteristics of Gene Reporter Systems (Click to enlarge)

Another usable reporter gene is the lacZ encoded enzyme β-galactosidase (1). Historically, this reporter gene has served as a valuable tool for cell and developmental biologists. The activity of β-galactosidase, comprised of a homotetramer of 116-kDa subunits, in a cell or fixed tissue can be monitored colorimetrically with its substrate 5- bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), which produces a blue precipitate upon cleavage. This enzyme's activity may also be detected fluorometrically in live cells with the synthetic substrate, fluorescein-β-digalactopyranoside (FDG) (2). Whereas the native substrate does not fluoresce, β-galactosidase cleavage of FDG produces free fluorescein that can be detected in live cells by epifluorescence microscopy or flow cytometry. Although the β-galactosidase/FDG combination has been used in FACS, it has shortcomings (3). First, because FDG is polar, it does not readily cross the plasma membrane. To introduce FDG inside cells, they must be subjected to a harsh hypotonic shock procedure, which leads to uneven substrate loading. Since fluorescence signal intensity from the cells depends on both enzyme and substrate concentrations, it is affected by uneven substrate loading. Second, the free fluorescein produced by FDG cleavage rapidly diffuses out of the cell. To minimize this efflux, cells must be kept on ice during the entire loading period. Currently, no other commercially available lipophilic substrate exists that can readily cross the cell membrane and quantitatively report the expression levels of β-galactosidase within a cell. In spite of the problems associated with the β-galactosidase/FDG system, it has been used as a tool for studying protein-protein interactions in live cells (4).