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| Current Opinion in Biotechnology Vol. 6, No. 1, February 1995 Marker proteins for gene expression [Review article] Keith V Wood Current Opinion in Biotechnology 1995, 6:50-58. |
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transforming
growth factor ;
The rapid progress in molecular genetics over the past two decades has expanded our ability to manipulate genetic structure, necessitating the development of methods for detecting and quantifying genetic activity. Methods for the direct measurement of gene expression include mRNA detection using oligonucleotide probes (northern blots) and protein detection using antibodies (western blots), but these methods are time consuming and thus costly. Furthermore, accurate quantification by these methods is typically limited as a result of technical restrictions. Reporter genes provide an alternative method of genetic analysis that in general, is more rapid and convenient.
The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological system, yields a readily measurable phenotype upon expression. This provides a convenient parameter that is correlated to the molecular events associated with genetic expression. The use of reporter genes today is so commonplace in molecular biology that they are now cited within virtually any journal issue in the current world literature.
Although new potential reporter genes are introduced each year, only a few
are used routinely. The most widely accepted reporter genes encode
chloramphenicol acetyltransferase (CAT), -galactosidase, -glucuronidase, and
firefly luciferase. Also commonly used are secreted alkaline phosphatase (SEAP)
and bacterial luciferase. A new reporter introduced this year, green fluorescent
protein (GFP), is attracting much interest because of its ability to
autocatalytically generate a fluorophore without addition of exogenous substrate
[1].
The measurable phenotype of these reporters is assayed by radioisotopes, color,
fluorescence, and luminescence; other reporters possess multiple assayable
phenotypes.
Because the range of reporters and their applications is so broad, a comprehensive review of this topic covering the past year is impractical. Instead, I focus on examples of the recently published research that are representative of the range of strategies employing reporter genes. I also briefly describe some of the distinguishing criteria used in selecting an appropriate genetic reporter, focusing especially on measurement sensitivity and reporter dynamics. Much of this discussion is drawn from my own experience in developing firefly luciferase as a genetic reporter.
Early applications of reporter genes focused primarily on analysis of cis -acting genetic elements, usually promoters and enhancers. Today, this is still the most common application for genetic reporters; however, the scope of this research has expanded. Although, most of this research is carried out, as before, using cells grown in culture, an increasing amount of work is employing transgenic animals and plants. For example, Lee et al. [2] have used transgenic mice to analyze the effects of mutations in different cis -regulatory elements of a cardiac gene promoter. In another report, efficient regulation of expression of genes introduced in the heart muscle of adult rats has been demonstrated through the incorporation of tetracycline in their diet [3]. Moreover, Fenerjian and Kafatos [4] have used two reporter genes to analyze a bidirectional promoter in transgenic Drosophila.
The spatial organization of gene expression in plants and animals is now
commonly analyzed using markers, most notably, -glucuronidase or
-galactosidase,
which deposit a colored or fluorescent indicator in expressing tissues [4][5][6][7].
Bylund et al. [8]
have even been able to spatially analyze gene expression within Bacillus
cells during spore formulation. Even so, these genetic staining methods in
general, disrupt cellular physiology, and interest currently centres on
measuring gene expression in intact living cells and organisms. The luciferases,
and more recently GFP, provide a means of measuring reporter activity in living
tissues without apparent stress on the cells. The luminescence from cells
expressing luciferases can be measured non-invasively using sensitive
charge-coupled device (CCD) cameras [9][10].
Luciferases in living cells have also been shown to provide dynamic measurements
of gene expression [11][12];
however, dynamic analysis has not yet been shown for GFP.
Studies on gene expression conventionally emphasize the DNA sequences defining transcriptional regulation. But as our understanding of molecular genetics has expanded, our view of the 'genetic event' has broadened accordingly to include the entire process of physiology regulation and phenotype expression. As a result, our use of genetic reporters has expanded from the analysis of cis -acting elements to the study of downstream events, such as RNA processing and protein synthesis, and upstream events, such as the biochemical mechanisms preceding DNA transcription. Reporter genes are capable of indicating events throughout the entire genetic process because their measurable parameter is an enzymatic phenotype. Usually, experimental conditions are established such that events other than transcriptional regulation are presumed to be constant. Changes in reporter expression are thus coupled to differences in transcriptional activity. Even so, alternative experimental strategies can reveal other stages within the broader genetic event.
Oliveira et al. [13] have used reporter genes to examine the role of stem-loop forming structures in the 5' untranslated region (UTR) of mRNA in yeast cells. In similar work, the same group has examined translational regulation in yeast by human iron-regulatory factor (IRF) on the iron-responsive element (IRE) located in the 5' UTR [14]. The role of 3' UTR structure in plant viral mRNA has been examined by Gallie and Kobayashi [15] in carrot protoplasts. Messenger RNA processing has been studied by Norris et al. [16] using the polyubiquitin genes in Arabidopsis to examine the effect of intron splicing on gene expression. In the human T-cell leukemia virus type 2 (HTLV-2), the RNA sequences promoting ribosomal frameshifting to yield the gagpro and gagpropol fusion genes have been investigated by Kollmus et al. [17]. The use of reporter genes to study protein synthesis is focused mainly on the role of chaperones in protein folding. The luciferases are particularly suitable for this purpose because they are relatively unstable, and their activity is instantaneously measurable upon refolding. Schroder et al. [12] have demonstrated the importance of chaperones for folding luciferase in Escherichia coli both in living cells and in cell extracts.
One of the most rapidly growing areas of reporter applications is the
analysis of transcription factors and intracellular signaling mechanisms that
underlie the regulation of DNA transcription. Jones et al. [18]
have used luciferase reporter genes to show that transcriptional activation by
the thyroid hormone receptor can be modulated by intracellular phosphatase and
kinase inhibitors. Luciferase has also been exploited to monitor the activity of
a temperature-sensitive mutant of p53 [19].
Park et al. [20] have
investigated the transactivation of virulence genes by PrfA in Listeria
monocytogenes using bacterial luciferase genes. Reporter genes may also be
used to elucidate undefined regulatory mechanisms. For example, Mifflin and
Cohen [21]
have studied stress response in cells injected with denatured proteins using the
-galactosidase gene
coupled to an hsp70 promoter. In another study, Gurvitz et al. [22]
have used a sporulation-specific promoter to identify mutants in the sporulation
regulatory pathway.
The effect of extracellular signals on gene regulation is also widely studied
using reporter genes. In a recent example, Himmler et al. [23]
have carried out a functional analysis of human dopamine receptors using firefly
luciferase. They found that dopamine analogs that interact with receptors in
transgenic cells modulate intracellular cAMP, which in turn regulates luciferase
expression through tandem cAMP-responsive elements (CREs). This research shows
the utility of reporter system for monitoring agonist and antagonist effects on
receptor activity in living cells. In analogous studies on human adenosine
receptors, Castanon and Spevak [24]
have shown that the same genetic construct of luciferase is effective when
applied to the analysis of different receptor types. Similar strategies have
been employed to measure receptor interactions with luteinizing
hormone/choriogonadotropin (LH/CG) [25],
transforming growth factor- (TGF-) [26],
antimineralocorticoids [27],
and a range of other steroid hormones [10].
Luciferase has also been used to assess androgen receptor function in clinical
tissue samples in a study of abnormalities in male sexual development [28].
The ability to couple reporter expression with extracellular factors has enabled the development of genetic biosensors. The studies above are examples of the use of markers as sensors for cellular growth factors, and in many cases, these sensors surpass the capabilities of alternative bioassay methods. In bacterial cells, this strategy is being used to measure environmental toxins. Heitzer et al. [29] have used bacterial luciferase in Pseudomonas to measure environmental naphthalene and salicylate bioavailability. Van Dyk et al. [30] have detected a range of environmental pollutants using heat-shock promoters in E. coli . Reporter genes can also be used as biosensors of active viruses. For example, Olivo et al. [31][32] have developed indicator cell lines that specifically identify infection by herpes simplex virus or by positive-strand RNA viruses.
In addition to their use as indicators of genetic activity, reporter genes
are also employed as genetic markers of specific tissues or organisms. A method
termed enhancer trapping can be used in transgenic organisms to identify tissues
with common genetic regulatory controls. Using this method, Callahan and Thomas
[33]
show how a tau-galactosidase
fusion reporter can be used to label different classes of neuronal cells in
Drosophila . The tau fusion allows visualization of cellular extension,
particularly of neuronal axons, which are not usually detectable using -galactosidase
alone. To measure the kinetics of human immunodeficiency virus (HIV) infection
in cell culture, Chen et al. [34]
have constructed an HIV virus containing the firefly luciferase gene. Reporter
genes are commonly used to measure the growth, ecology, and pathogenicity of
bacteria [35].
For these applications, the most popular reporters are -galactosidase and
bacterial luciferase.
Finally, reporter genes are widely used as markers in the development of
genetic transformation methodologies. Examples include the development of
transferrin-mediated transfection of mammalian cells [7],
transfection of mammalian cells by particle bombardment [36],
and lipospermadine-based transfection of vertebrate embryos [6]. An
area of great interest is the use of markers to assess the effectiveness of
gene-therapy technology. Gal et al. [37]
have studied myocardial transfection in rabbits and microswine by direct
injection of the firefly luciferase gene into cardiac muscle. In another study,
Mazur et al. [38]
have used adenovirus to introduce luciferase and -galactosidase into
porcine coronary arteries.
For most purposes, the primary consideration in selecting a reporter gene is convenience. Measurements of gene expression can often be performed through direct assay of specific mRNAs, but this involves substantially greater effort. Reporter genes provide analogous information much more efficiently. Even so, convenience is a matter of one's own particular circumstances and includes a consideration of available equipment and reagents, specific experimental objectives, and experience with the reporter in the biological system.
The acetylation reaction of CAT is usually measured using 14
C-chloramphenicol or 3 H-acetyl-CoA and thus requires a scintillation
counter for quantitation [4][39][40].
For most researchers, the use of these isotopes is becoming more problematic
because of more rigid restrictions on waste disposal. A fluorescent assay of CAT
activity is now available, but not widely used. Enzymatic activity of -galactosidase, -glucuronidase, and
alkaline phosphatase can be measured by colorimetric [41],
fluorescent [42], or
luminescent assays [43];
thus, quantitation may be achieved by several means. Luciferase activity ideally
is quantitated using a luminometer [10][14][25][27][44];
however, scintillation counters and sensitive fluorometers may also be used. GFP
is quantitated using a fluorometer. For spatial analysis of gene activity,
detection of color [4][6][33] or
fluorescence [8]
deposition by -galactosidase or
-glucuronidase is
most common, although recently, much enthusiasm has surrounded the use of GFP [1].
Spatial detection of luciferases is usually achieved using sensitive CCD photon
detectors [9][10][11][44].
Photographic film may be used in some cases, but often it does not provide
sufficient sensitivity [44].
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Fig. 1.Schematic representation of luminescent and fluorescent reporter assays. The vertical dimension represents relative photon flux. The bars show the approximate range of photon flux supported by different reporter types in purified form. The shaded region of each bar represents the approximate range of interference expected from endogenous activities in biological systems. The approximate sensitivity limits for different photon detection methods are also illustrated. The diagram only roughly illustrates the relationship between different reporter technologies and detection methods; actual ranges of reporter performance and instrument sensitivities depend greatly on specific circumstances. |
Sensitivity has always been an important criteria of reporter performance; however, it is not always properly evaluated. It is typically equated with signal strength, without proper regard for background and assay precision. The assay of reporter activity is a measure of signal (s) over background (b), (i.e. s - b). If the determinations of s and b were infinitely precise, then the reporter assay would be infinitely sensitive. In reality, all measurements have associated error and thus a more applicable expression is (s ± error) - (b ± error), or (s - b) ± (cumulative error). The limit in sensitivity is reached when the difference between signal and background is about equal to the assay precision. Because precision is typically proportional to signal magnitude (e.g. value ± percent error), a greater background has a greater associated error, and thus a greater limit on assay sensitivity.
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Fig. 2.Model system showing the relationship between reporter stability and dynamic response. The levels of stable reporter (t 1/2= 50 h) and unstable reporter (t 1/2= 3 h) are shown, together with the relative rate of transcription. (a)Comparison of reporter expression (arbitrary units). (b)Comparison of relative reporter expression (normalized to maximum expression). (c)Relative reporter expression with circadian control of gene regulation. |
The difference between signal and background is dependent on the reporter chemistry, instrument sensitivity, and interfering chemical activities. The effect of these parameters can be illustrated by comparing reporter assays that are based on photon flux (Fig. 1). Because the enzymatic turnover of chemiluminescence-based reporters is typically greater than luciferases, they yield a greater signal strength (i.e. photon flux). Moreover, the signal strength of fluorescence, which is proportional to the photon flux of the excitation light source, is greater than all of the luminescence chemistries. Nevertheless, the background of luciferase assays is limited only by the sensitivity of the luminometer. Chemiluminescence yields a measurable photon flux in the absence of enzyme, and thus is limited by the chemical mechanism. When assaying purified enzymes, chemiluminescence and bioluminescence methods typically have comparable sensitivities.
Endogenous enzymatic activities present in most biological systems, however, significantly increase the background of the chemiluminescent-based reporters. No analogous endogenous activity exists for the luciferases, so in reporter applications, the signal to background difference is generally greater for bioluminescence. This difference, though, also depends on the detection method. For instance, in photographic detection, where the background of all luminescence-based reporters is limited by the sensitivity of the film, the greater photon flux of chemiluminescence yields a greater signal over background. The limitation of endogenous activity is even more limiting for fluorescence-based reporters because of the abundance of background fluorescence in biological systems. Even for GFP, which lacks endogenous homologs in most systems, the prevalence of other fluorescent molecules greatly limits sensitivity. Nevertheless, the very high photon flux of fluorescence makes such reporters useful when using low-sensitivity detection methods. This explains why GFP is more useful than firefly luciferase in fluorescence microscopy, even though bioluminescence generally is much more sensitive than fluorescence.
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Fig. 3.Circadian expression of luminescence in transgenic Arabidopsis. Firefly luciferase expression driven by the cabpromoter (for the gene encoding chlorophyll a/bbinding protein) is assayed by addition of luciferin to seedlings grown in microtiter dishes. The seedlings were measured repeatedly over approximately ten days in constant light. |
Because of the widespread importance of genetic reporters in molecular genetics, every year witnesses the introduction of new improvements to the assay methods. Most of these refinements are directed at making the assays more sensitive, often through additional processing to minimize endogenous interferences [39][40][41][42]. Although these modifications bring benfits (varying degrees), the additional manipulations required increase the overall effort necessary to perform the reporter assays. Furthermore, more complex protocols probably also yield greater cumulative error, which can offset gains made in the signal to background difference. In most research environments, assay simplicity is an important characteristic for achieving consistently reproducible results. Other important characteristics are linearity and reliability. All the commonly used reporters have been demonstrated to be generally reliable; firefly luciferase has a particularly large linear range (over 10 8 -fold) and the assay requires less than 1 min to complete.
The ability of a reporter to indicate changes in gene expression (i.e. the dynamic response of a reporter) is a property that is often taken for granted. For instance, enzymatic stability is commonly proclaimed beneficial to reporter performance without consideration for its inverse relationship to dynamics [44]. To respond rapidly to changes in gene expression, the genetic reporter must have a short half-life within the biological system. This can be illustrated with a simple model where reporter synthesis is proportional to gene transcription (i.e. zero-order kinetics, k 1 ) and reporter degradation is proportional to its concentration (i.e. first-order kinetics, k 2 ). Thus, changes in the reporter concentration (dC) can be described by dC = (k 1 + k 2 C)dt. It has been estimated that the half-life (ln[0.5]/k 2 ) of firefly luciferase and CAT in mammalian cells is 3 h and 50 h, respectively, although these can vary significantly in different hosts [45]. These values can be used in the model to show how luciferase and CAT are expected to respond to a rapid change in gene expression (Fig. 2).
At first appearance, CAT is apparently more responsive to a change in gene expression than luciferase (Fig. 2a). Reporter assays are, however, interpreted by the relative change in expression (Fig. 2b). Such an analysis reveals that luciferase actually responds much more rapidly than CAT. In an extreme case of genetic regulation through a circadian mechanism (Fig. 2c), the luciferase model is able to reveal the cyclic pattern of gene expression, whereas CAT exhibits an almost constant signal. This prediction is supported by data from an analysis of the circadian-controlled cab promoter in Arabidopsis (Fig. 3c). Luciferase activity clearly shows cyclic regulation of this promoter over a nine day period; in contrast, this regulation pattern is not revealed using CAT [11].
The choice of an appropriate reporter may depend on specific characteristics
of the biological system under study. The greatest concern is, in general, the
level of interfering endogenous activity. For this reason, -glucuronidase is
not commonly used in mammalian cells nor is -galactosidase used
in plant cells. Firefly luciferase is widely employed in most experimental
systems because no endogenous bioluminescence is present. Because bacterial
luciferase is a dimeric protein, it cannot be expressed from a single gene and
thus is generally limited to bacterial systems. Fusion forms are available, but
they do not perform as well as firefly luciferase in eukaryotic systems.
Unexpected interactions may occur with any reporter in a complex biological
system, and periodically, reports warn of potential limitations under specific
conditions [5][46][47][48].
To avoid potential interference caused by the native translocation of luciferase
into peroxisomes, my colleagues and I have recently developed a cytoplasmic form
of this enzyme. We have also engineered several other modifications to increase
the general utility of this reporter [49].
Reporter genes are widely used in a diverse range of applications, and each year, the scope of this range broadens. In the past year, applications have continued to be reported in the analysis of genetic events upstream and downstream of DNA transcription, and further emphasis has been given to in vivo analysis in transgenic animals and plants. Future trends will show an increased focus on the genetic analysis of individual cells and on non-invasive analysis methods. This will involve further refinement of both the chemistries and instrumentation of reporter analysis.
The past year has also introduced new reporters and new improvements to
reporter assays. The most promising new reporter is GFP, which is a non-invasive
fluorescent indicator of gene expression [1].
This year has also witnessed the expansion of chemiluminescence-based reporter
assays to include -glucuronidase [43].
The trend toward the adoption of luminescent and fluorescent reporter assays
will probably continue into the future because of the sensitivity and range of
these methods [44][50].
The choice of system, from an ever-broadening range of available reporter systems, also entails consideration of a greater number of performance criteria. Present technology is sufficiently advanced that, for most applications, assay convenience is the major consideration. Assay reliability is also important, but in general, is not a problem for the most commonly used reporters, with the exception of idiosyncratic behavior in specific biological systems. Assay sensitivity must be sufficient to meet experimental objectives, and is determined by the assay chemistry, instrument sensitivity, and interfering chemical activities. Also relevant are assay linearity and simplicity and, in many circumstances, reporter dynamics. The advances made in reporter versatility and performance reflect the general importance of this technology to biological analysis.
I thank Dr Steve Kay, of the University of Virginia, for use of his data to illustrate dynamic expression of firefly luciferase.
-galactosidase and
CAT activity are used together to analyze a bidirectional promoter
inDrosophila melanogaster. Mutations within the bidirectional promoter
abolish expression from both genes.-galactosidase
gene fails to yield similar results. The authors suggest that the -galactosidase
gene may exert a cis effect on expression in transgenic mice.-galactosidase and
firefly luciferase expression to develop a method for introducing genes into
chick embryos by lipospermadine-based transfection. The cationic lipid
Transfectam tmwas used to transfect the reporter gene
generally, or to target the gene locally through microinjection. Quantitative
analysis of the method is carried out using luciferase; -galactosidase is
used for determination of spatial expression.-galactosidase
reporter gene. Expression of -galactosidase
activity is visualized by fluorescence microscopy using a fluorogenic
substrate. Through the use of different promoters, gene expression can be
clearly seen either throughout the cell, in the prespore region, and in the
forespore.-glucuronidase and
firefly luciferase. Using electroporation of in vitro synthesized RNA
constructs, differences in mRNA stability and translation efficiency are
measured.-glucuronidase and
luciferase activity to analyze the effect of these introns on
expression.-galactosidase and
firefly luciferase genes. Without frameshifting, only -galactosidase
activity is detectable; with frameshifting, a -galactosidaseluciferase
fusion is synthesized that exhibits luminescence activity. The efficiency of
frameshifting is determined as the ratio of luminescence to -galactosidase
activity. Expression of -galactosidase and
luciferase are correlated with immunoreactivity in western blots.-galactosidase
activity from a heat-shock promoter. The results show that stress response is
dependent on the mode of protein denaturation and the location of
intracellular injection.-galactosidase
reporter gene is coupled to a sporulation-specific promoter to provide a clear
phenotype during the sporulation process. From this phenotype, three classes
of sporulation mutations are isolated: those which overexpress the reporter
gene under sporulation conditions, those which do not express the gene under
any condition, and those which express the gene in vegetative cells not
undergoing sporulation. using cells
transfected with a plasminogen activator inhibitor-1 promoter-luciferase
construct. is a potent
regulator of cellular differentiation, proliferation, migration, and protein
expression. In this paper, a bioassay for TGF- is developed
using stably transformed cells containing the firefly luciferase gene coupled
to a plasminogen activator inhibitor-1 promoter. The cell line yields
dose-dependent luminescence to TGF- in the range of
0.2 mM to >30 mM, providing greater sensitivity and specificity than widely
used alternative bioassays.-galactosidase, an
axon-targeted fusion protein.-galactosidase is
limited by its inability to readily diffuse into axons. In this paper, a
modified form of the enzyme is constructed by fusing the cDNA encoding bovine
microtubule-binding protein, tau, onto the -galactosidase
gene. Using an enhancer-trap transposon inDrosophila , this modified
reporter is used to mark various neuronal cell types, as well as muscle fibers
and glial cells.-galactosidase,
catechol 2,3-dioxygenase (encoded byxylE), 2,4-dichlorophenoxyacetate
monooxygenases, and bacterial luciferases. The use of bacterial luciferases
constitutes the greatest part of the review. Detection methods for
luminescence are also discusssed.-galactosidase is
used to visualize the spatial pattern of particles in cell-culture dishes. The
particle bombardment method is compared with transformation by
electroporation, lipofection, and diethylaminoethyl dextran.-galactosidase
reporter genes.-galactosidase in
mammalian cells and its application in assays of eukaryotic promoter
activity.-galactosidase as
a reporter gene are described. The thermostable reporter is cloned from the
thermoacidophilic archaebacteriumSulfolobus solfataricus. The reporter
exhibits very little activity at 37°C; enzyme activity is assayed by
incubation at 75°C. A preliminary comparison with CAT is also given.-galactosidase in
assays for inhibitors of HIV-1 TAT using bacterial -galactosidase as
a reporter enzyme.-galactosidase
assay by reducing interference from endogenous enzymatic activity. Heat
treatment at 50°C for 1 h inactivates the -galactosidase
activity endogenous to several eukaryotic cell lines by as much as 40-fold
without adversely affecting the activity of bacterial -galactosidase.-glucuronidase
using adamantyl dioxetane derivatives.-galactosidase
yields expected results. Coupling of bacterial luciferase to thelac
promoter or the gyrB promoter does, however, report expected gene expression
patterns.
