Detection of β‐lactamase reporter gene expression by flow cytometry

Background: Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes. This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest. The most commonly used reporters are green fluorescent protein and β‐galactosidase (lacZ). In this study, we characterized the performance of a cell‐permeant, ratiometric, β‐lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM). We compared its characteristics with that of the β‐galactosidase/fluorescein di‐β‐D‐galactopyranoside reporter system. Methods: Jurkat cell lines were generated for β‐lactamase and β‐galactosidase reporters with the use of similar plasmid constructs. Rare event flow cytometric detection for the β‐galactosidase and β‐lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter. To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time. Technical issues related to data processing and optical configuration are also presented. Results: The low population coefficients of variation afforded by ratiometric detection of the β‐lactamase system improved the statistical performance of the assay in comparison with a single‐dye, intensity‐based assay, leading to markedly improved detection for low copy number and rare events. At low levels of gene expression, β‐lactamase was detected with approximately 10‐fold higher confidence than was β‐galactosidase. In rare event detection experiments, cells expressing high levels of β‐lactamase proteins were reliably detected at frequencies of 1:106 compared with about 1:104 for β‐galactosidase. Conclusion: The ratiometric fluorescence readout of the β‐lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available β‐galactosidase substrates. Further, the cell‐permeant nature of the substrate improved experimental convenience. These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low‐expressing or downregulated genes. Cytometry Part A 51A:68–78, 2003. © 2003 Wiley‐Liss, Inc.