Protocols and Applications Guide (US Letter Size) - Promega

|||||||| 8Bioluminescence Reporters
The speed by which a genetic reporter can respond to
changes in the transcriptional rate is correlated to the
stability of the reporter within cells. Highly stable reporters
accumulate to greater levels in cells, but their concentrations
change slowly with changes in transcription. Conversely,
lower stability yields less accumulation but a much faster
rate of response. To provide reporters designed to meet
different experimental needs, families of luciferase genes
have been developed yielding different intracellular
stabilities. The genes conferring lower stabilities are referred
to as the Rapid Response Reporters.
Beetle and Renilla luciferase reporters have an intrinsic
protein half-life of ~3 hours. However, reporter response
may still lag behind the underlying transcriptional events
by several hours. To further improve reporter performance,
we have developed destabilized luciferase reporters by
genetically fusing a protein degradation sequence to the
luciferase genes (Li et al. 1998). After evaluation of many
degradation sequences for their effect on response rate and
signal magnitude, two sequences were chosen, one
composed of the PEST protein degradation sequence and
a second composed of two protein (CL1 and PEST)
degradation sequences. Due to their increased rate of
degradation, these destabilized reporters respond faster
and often display a greater magnitude of response to rapid
transcriptional events and are therefore called the Rapid
Response Reporters.
Vector Backbone Design
Vectors that are used to deliver the reporter gene to the
host cells are also critical for the overall performance of the
reporter assay. Cryptic regulatory sequences such as
transcription factor binding sites and/or promoter modules
found on the vector backbone could lead to high
background and anomalous responses. This is a common
issue for mammalian reporter vectors including our pGL3
Luciferase Reporter Vectors, which have recently been
improved. We have extended our successful "cleaning"
strategy for reporter genes to the entire pGL3 Vector
backbone, removing cryptic regulatory sequences wherever
possible, while maintaining functionality. Other
modifications include a redesigned multiple cloning region
to facilitate easy transfer of the DNA element of interest,
removal of the f1 origin of replication and deletion of an
intronic sequence. In addition, a synthetic poly(A)
signal/transcriptional pause site was placed upstream of
either the multiple cloning region (in promoterless vectors)
or the HSV-TK, CMV or SV40 promoter (in
promoter-containing vectors). This extensive effort resulted
in the totally redesigned and unique vector backbone of
the pGL4 Luciferase Reporter Vectors.
The pGL4 family of luciferase vectors incorporates a variety
of features such as a choice of luciferases, Rapid Response
versions, mammalian-selectable markers, basic vectors
without promoters and promoter-containing control vectors
(Figure 8.5).
Protocols & Applications Guide
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rev. 3/09
By manipulating luciferase genes we’ve developed a series
of optimized reporter genes featuring additional
luminescence colors and improved codon usage, while
deleting cryptic regulatory sequences such as transcription
factor binding sites that could decrease protein expression
in mammalian cells. The following section provides
information about specific bioluminescence reporters and
assays, including how to choose the correct reporter genes
and vectors to suit your research needs.
Advantages of the pGL4 Luciferase Reporter Vectors
1. Improved sensitivity and biological relevance due to:
• Increased reporter gene expression: Codon
optimization of synthetic genes for mammalian
expression
• Reduced background and risk of expression
artifacts: Removal of cryptic DNA regulatory
elements and transcription factor binding sites
• Improved temporal response: Rapid Response
technology available using destabilized luciferase
genes
2. Additional advantages include:
• Flexible detection options: Choice of reporter genes
• Easy transition from transient to stable cells: Choice
of mammalian selectable markers
• Easy transfer from one vector to another: Common
multiple cloning site and a unique SfiI transfer
scheme
III. Luciferase Reporter Assays and Protocols
The challenge for designing bioluminescence assays is
harnessing this efficient light-emitting chemistry into
analytical methodologies. Most commonly this is done by
holding the reaction component concentrations constant,
except for one component that is allowed to vary in relation
to a biomolecular process of interest. When the reaction is
configured properly, the resultant light is directly
proportional to the variable component, thus coupling an
observable parameter to the reaction outcome. In assays
using luciferase, the variable component may be the
luciferase itself or its substrates or cofactors. Because of
very low backgrounds in bioluminescence, the linear range
of this proportionality can be enormous, typically extending
104- to 108-fold over the concentration of the variable
component.
Choosing the assay appropriate for your research needs is
assisted by the following considerations and Tables 8.1 and
8.2, showing available luciferase genes, assays and reagents.
A. Single-Reporter Assays
Assays based on a single reporter provide the quickest and
least expensive means for acquiring gene expression data
from cells. However, because cells are inherently complex,
the quantity of information gleaned from a single-reporter
assay may be insufficient for achieving detailed and
accurate results. Thus one of the first considerations in
choosing a reporter methodology is deciding whether the
speed and depth of information from a single reporter is
PROTOCOLS & APPLICATIONS GUIDE 8-7