Protocols and Applications Guide (US Letter Size) - Promega

|||||||| 8Bioluminescence Reporters
In a system where a second reporter is used, a "control"
vector can be used to normalize for transfection efficiency
or cell lysate recovery between treatments or transfection
experiments. Typically, the control reporter gene is driven
by a constitutive promoter and is cotransfected with
"experimental" vectors. The experimental regulatory
sequences are linked to a different reporter gene so that the
relative activities of the two reporter gene products can be
assayed individually. Control vectors can also be used to
optimize transfection methods. Gene transfer efficiency is
typically monitored by assaying reporter activity in cell
lysates or by staining the cells in situ to estimate the
percentage of cells expressing the transferred gene.
In general, bioluminescence reporters are preferred when
experiments require high sensitivity, accurate quantitation
or rapid analysis of multiple samples.
Dual-bioluminescence assays can be particularly useful for
efficiently extracting information.
II. Luciferase Genes and Vectors
A. Biology and Enzymology
Bioluminescence as a natural phenomenon is widely
experienced with amazement at the prospect of living
organisms creating their own light. Basic research into this
phenomenon has also led to practical applications,
particularly in molecular biology where bioluminescence
enzymes have been widely used as genetic reporters.
Moreover, the value of this application has grown
considerably over the past decade as the traditional use of
reporter genes has broadened to cover wide ranging aspects
of cell physiology.
The conventional use of reporter genes has been largely to
analyze and dissect the function of cis-acting genetic
elements such as promoters and enhancers (so-called
"promoter bashing"). In typical experiments, deletions or
mutations are made in a promoter region, and their
consequential effects on coupled expression of a reporter
gene are then quantitated. However, the broader aspect of
gene expression entails much more than transcription alone,
and reporter genes can also be used to study these other
cellular events.
Some examples of analytical methodologies that use
luciferase include:
• Stable cell lines that integrate the reporter gene of
interest into the chromosome can be selected and
propagated when a selectable marker is included in a
transfection vector. These types of engineered cell lines
have been used for drug screening and to monitor the
effect of exogenous agents and stimuli upon gene
expression.
• Identification of interacting pairs of proteins in vivo
using a system known as the two-hybrid system (Fields
et al. 1989). The interacting proteins of interest are
brought together as fusion partners—one is fused with
a specific DNA binding domain, and the other protein
is fused with a transcriptional activation domain. The
physical interaction of the two fusion partners is
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necessary for the functional activation of a reporter
gene driven by a basal promoter and the DNA motif
recognized by the DNA binding protein. This system
was originally developed with yeast but has also been
used in mammalian cells.
• Bioluminescence resonance energy transfer (BRET) for
monitoring protein-protein interactions, where a fusion
protein is made using the bioluminescent Renilla
luciferase and another protein fused with a fluorescent
molecule. Interaction of the two fusion proteins results
in energy transfer from the bioluminescent molecule
to the fluorescent molecule, with a concomitant change
from blue light to green light (Angers et al. 2000).
Luciferase genes have been cloned from bacteria, beetles
(e.g., firefly and click beetle), Renilla, Aequorea, Vargula and
Gonyaulax (a dinoflagellate). Of these, only the luciferases
from bacteria, beetles and Renilla have found general use
as indicators of gene expression. Bacterial luciferase,
although the first luciferase to be used as a reporter, is
generally used to provide autonomous luminescence in
bacterial systems through expression of the lux operon.
Ordinarily it is not useful for analysis in mammalian cells.
Firefly Luciferase
Firefly luciferase is by far the most commonly used
bioluminescent reporter. This monomeric enzyme of 61kDa
catalyzes a two-step oxidation reaction to yield light,
usually in the green to yellow region, typically 550–570nm
(Figure 8.1). The first step is activation of the luciferyl
carboxylate by ATP to yield a reactive mixed anhydride.
In the second step, this activated intermediate reacts with
oxygen to create a transient dioxetane that breaks down to
the oxidized products, oxyluciferin and CO2. Upon mixing
with substrates, firefly luciferase produces an initial burst
of light that decays over about 15 seconds to a low level of
sustained luminescence. This kinetic profile reflects the
slow release of the enzymatic product, thus limiting
catalytic turnover after the initial reaction (Figure 8.1).
Various strategies to generate a stable luminescence signal
have been tried to make the assay more convenient for
routine laboratory use. The most successful of these
incorporates coenzyme A to yield maximal luminescence
intensity that slowly decays over several minutes. The
mechanism of action for coenzyme A in the luminescent
reaction is unclear, although it probably stems from the
evolutionary ancestry of firefly luciferase. The amino acid
sequence of firefly luciferase is related to a diverse family
of acyl-CoA synthetases. By analogy to the catalytic
mechanism of these related enzymes, formation of a thiol
ester between CoA and luciferin seems likely. An optimized
assay containing coenzyme A generates relatively stable
luminescence in less than 0.3 seconds with linearity to
enzyme concentration over a 100-millionfold range. The
assay sensitivity allows quantitation to fewer than 10–20
moles of enzyme.
The popularity of native firefly luciferase as a genetic
reporter is due both to the sensitivity and convenience of
the enzyme assay and to the tight coupling of protein
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