15] Fusions to β-lactamase as a reporter for gene expression in live mammalian cells

Abstract

This chapter discusses the role of β-lactamase as a reporter enzyme for monitoring gene expression in mammalian cells. The β-lactamase enzyme used as a reporter in mammalian cells is a member of a large and structurally diverse family of enzymes that cleave β-lactam antibiotics, such as penicillins and cephalosporins. β-lactamase enzyme is of bacterial origin without any mammalian homologues and permits the quantitative analysis of reporter expression in single mammalian cells without interference from an endogenous background. It can be fused to the N and C termini of proteins without loss of activity, which allows monitoring the expression levels of the tagged protein. It is distinguished from popular luciferase and β-galactosidase reporter enzymes in that it can be detected sensitively in individual living cells under standard culture conditions. This is accomplished with a fluorogenic membrane-permeable substrate ester that diffuses into cells and provides high intracellular concentrations of accumulated fluorescent substrate. The reporter is detected sensitively by its catalysis of substrate hydrolysis, which results in a highly amplified fluorescent signal, allowing analysis of expression of proteins that are present in cells at low copy numbers. The fluorescent signal from live cells permits a facile functional selection of reporter-expressing cells with flow cytometry or fluorescence microscopy. Selected cells usually remain viable and can be used for the subsequent analyses that require intact cells.