News | April 26, 1999

Live and in Color: Monitoring Gene Expression in Real Time

DNA chips are all the rage these days—after all, how can you beat measuring the activity of thousands of genes at once? But gene expression is a dynamic process, and while it is possible to analyze multiple samples on chip arrays, doing kinetics that way can be cumbersome, not to mention costly, as most chips are designed for a single-use. Since the early 1990s, fluorescent probes—green fluorescent protein (GFP) in particular, and a number of variants derived from it—have come into wide use as monitors of gene expression in live cells. However, these probes suffer from being somewhat insensitive—requiring upwards of 30,000 molecules per cell in even the brightest variants—and too stable to monitor any rapid changes in gene expression. More recently, a catalytic, fluorogenic assay has been developed that combines sensitivity with the ability to monitor live, individual cells.

Mr. Green Genes
Keeping It Real

Mr. Green Genes (Back to Top)
Green fluorescent protein from the jellyfish Aequoria victoria is an intrinsically fluorescent protein that has found many uses in the imaging of biological processes. Among its attractive and useful features are:

  • It requires no co-factors or substrates, being intrinsically fluorescent.
  • It is stable, on the order of one day in living cells.
  • It retains fluorescence when fused to other proteins.
  • It can be expressed in a wide variety of species and cell types.

In addition, a number of variants of GFP have been isolated to make up for some of its shortcomings: GFPs with simpler excitation profiles have been described (the native protein has two peaks of excitation, which limits its usefulness in double label experiments), variants that fluoresce more brightly than the native protein are available, as are variants with base substitutions to optimize expression in heterologous systems. This collection of molecules has been extremely useful to monitor the translocation of proteins within cell compartments (Ref. 1, for example), and have also been used successfully as a reporter of gene activity (Ref. 2), and to visualize expression patterns in embryos (Ref. 3).

A number of companies offer vectors with GFP and various derivatives, among them Clontech (Palo Alto, CA), Display Systems Biotech (Vista, CA), Pharmingen (La Jolla, CA), and Quantum Biotechnologies (Montreal).

However, despite all the tinkering that has been done to the molecule, GFP and its descendants still are less than ideal for monitoring gene expression changes in real time. Even the brightest variants still require on the order of 104 and 105 fluorescent molecules to be detected, leaving it out of the running for low abundance proteins, which are the majority of proteins, if not also the most interesting. In addition, the long half-life of the protein makes it unusable for looking at cyclic or rapid changes in gene expression. A "destabilized" variant sporting the PEST domain from mouse ornithine decarboxylase has been engineered (and marketed by Clontech), the half-life of which has been reduced to several hours, still a bit long for some of the more dynamic processes.

Keeping It Real (Back to Top)
A FRET (fluorescence resonance energy transfer) based assay has been developed by researchers at Aurora Biosciences (San Diego) and the University of California, San Diego (UCSD), for monitoring real time gene expression in individual cells. This assay employs a catalytic process, and hence amplifies the signal, leading to a great improvement in sensitivity over direct fluorescence assays, like GFP. It also, as described below, uses ratio imaging, which provides internal normalization for a variety of factors that can lead to sample to sample variability.

Fluorescence resonance energy transfer is a process whereby a fluorescent molecule, upon excitation, transfers energy to a near-by molecule, rather than fluoresce itself. The acceptor molecule can either accept (i.e. fluoresce) or quench the fluorescence. It requires that the two molecules be within a certain distance, and therefore can be used to monitor reactions that would result in a change in the donor/acceptor spacing.

FRET is basis for molecular beacons, for example (Ref. 4). Here two fluorescent molecules are positioned at either end of an oligo probe that forms a stem and loop structure. Upon hybridization to a target sequence, the stem and loop structure is disrupted, changing the spacing between the donor and acceptor molecule, and resulting in the generation of a fluorescence signal. FRET probes have also been developed for real-time, quantitative PCR reactions, for sequencing reactions, and for the measurement of voltage-dependent changes in single cells (Ref. 5).

In the FRET-based gene reporter assay, ß-lactamase (that old workhorse ampicillin resistance gene, modified slightly to keep it inside cells) is used as a reporter gene in combination with a fluorogenic substrate designed by the group at Aurora. The researchers created a fluorogenic, membrane-permeable substrate for ß-lactamase, a cephalosporin derivative with two fluorophores attached. The substrate fluoresces green upon excitation with violet light (or UV) when intact, but upon cleavage with ß-lactamase, it shows a shift in fluorescence to the blue due to the loss of FRET. This assay, since it ratios a shift in fluorescence, has the additional advantage of enabling internal corrections for local changes in substrate concentration, differences in cell size, and slight variations in excitation and emission intensity.

Gregor Zlorarnik and his colleagues introduced the ß-lactamase assay in a 1998 Science paper entitled "Quantitation of Transcription and Clonal Selection of Single Living Cells with ß-lactamase as Reporter" (Ref. 6). Here they showed that the assay could easily detect 10–20,000 reporter molecules within an hour, and, with longer incubation times could detect as few as 50 copies of the reporter molecule per cell. In addition, looking at several cell lines that had been transfected with ß-lactamase attached to a hormone responsive element, they were able to quantitate the response to the agent in the population and in individual cells, revealing cell to cell heterogeneity. They also used their assay to measure the dose-response and the kinetics of transcription, and to isolate individual expressing cells by fluorescence-activated cell sorting.

And there's more. Several reports have come out recently in which this system has been used in interesting and novel ways. In an elegant study published in Nature last year (Ref. 7) Roger Tsien's group at UCSD, where much of the development of FRET probes has been done, used the ß-lactamase system to quantitate the effect of calcium oscillations on gene expression. Raz et al (Ref. 8) used the assay as a marker for gene expression in zebrafish embryos and, in direct comparison with a GFP, showed that the ß-lactamase assay is superior for detecting low levels or rapid changes in gene expression.

Finally, using ß-lactamase, the group at Aurora has developed a method for genome-wide screening for genetic elements that respond to stimuli—drugs, hormones, or other effectors (Ref. 9). This process, termed "gene tagging," introduces a promoterless ß-lactamase gene into a population of cells from which individual clones responding to a particular stimulus can be identified and cloned. The clones can be used to study the response in more detail, and ultimately to retrieve the gene through its association with the reporter ß-lactamase. The company believes that this system has the potential to speed the process of drug discovery—both by eliminating the need for costly and time-consuming cloning of cells and through the ability to measure responses in live, individual cells.

For more information on the ß-lactamase assay, contact Janice Corey at Aurora Biosciences Corp., 11010 Torreyana Road, San Diego, CA 92121, 619-404-6728.

References (Back to Top)

  1. Barak, L.S., S.G. Ferguson, J. Zhang, and M.G. Caron, J. Biol. Chem. 272:274–97, 1997.
  2. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher, "Green Fluorescent Protein as a Marker for Gene Expression," Science 263:802-805, 1994
  3. Prasher, D., "Using GFP to see the light," Trends Genet 1:320–323, 1995.
  4. Tyagi, S. and F.R. Kramer, "Molecular Beacons: Probes that Fluoresce upon Hybridization," Nature Biotechnol. 14:303–308, 1996.
  5. González, J. E. and R.Y. Tien, "Voltage Sensing by Fluorescence Energy Transfer in Single Cells," Biophys. J. 69:1272–80, 1995.
  6. Zlokarnik, G., P.A. Negulescu, T.E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney, K. Roemer, and R.Y. Tsien, "Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter." Science 279:84–8, 1998.
  7. Li, W., J. Llopis, M. Whitnet, G. Zlokarnik, and R. Tsien, "Cell-permeant caged InspP3 ester shows that Ca2+ spike frequency can optimize gene expression." Nature 392:938–941, 1998.
  8. Raz, E., G. Zlokarnik, R. Tsien, and W. Driever, "ß-lactamase as a marker for Gene Expression in Live Zebrafish Embryos," Developmental Biology 203:290–294, 1998.
  9. Whitney, M., D. Rockenstein, G. Cantin, T. Knap, G. Zlokarnik, P. Sanders, K. Durick, F.F. Craig, and P.A. Negulescu, "A genome-wide functional assay of signal transduction in living mammalian cells," Nature Biotechnology 16:1329–1333, 1998.

By Laura DeFrancesco