New reporter plasmids increase the versatility of the
PathDetect® trans-reporting
systems
Assess Activation of Signal Transduction Pathways Using Choice
of Reporter Proteins
Li Xu • Fannie Chau • Tim Sanchez • Mary Buchanan •
Chao-Feng Zheng
Four new reporter plasmids are available for the
PathDetect ®
trans-reporting systems. These encode the researcher’s choice of
b-galactosidase (ß-gal), secreted alkaline phosphatase
(SEAP), chloramphenicol acetyltransferase (CAT), or humanized
Renilla green fluorescent protein (hrGFP) reporter
genes.*,
With these
reporter plasmids, researchers can detect the activation of specific
signaling pathways by assaying for ß-gal, SEAP, CAT, or hrGFP enzyme reporters
in addition to the original luciferase reporter. We tested and determined
that these new trans-reporter plasmids were as effective for readout of
activation as the luciferase plasmid.
When Stratagene introduced the
PathDetect in vivo signal transduction pathway systems1 in 1997, it rapidly became popular
within the flourishing field of functional genomics. To measure gene
activity using transduction pathways, a fusion trans-activator
plasmid is cotransfected into mammalian cells with a reporter plasmid and
an uncharacterized gene. The uncharacterized gene product may then
directly or indirectly phosphorylate the fusion trans-activator
protein, thus activating transcription of the reporter gene from the
reporter plasmid. If the reporter protein activity increases above
background levels, it shows that the gene of interest is involved in the
pathway being evaluated.
Table 1
Pathways Available
Pathway
Transcriptional Activator
PathDetect® System
Plasmids available
JNK (c-Jun N-Terminal Kinase)
c-Jun
PathDetect® c-Jun Trans-Reporting
System
pFA2-cJun plasmid
pFC-MEKK plasmid
MAPK (Mitogen-Activated Protein Kinase)
and JNK
Elk1
PathDetect® Elk1 Trans-Reporting System
pFA2-Elk1 plasmid
pFC-MEK1 plasmid
PKA
(Cyclic AMP-Dependent Protein Kinase)
CREB
PathDetect® CREB Trans-Reporting System
pFA2-CREB plasmid
pFC-PKA plasmid
p38 MAP Kinase
CHOP
PathDetect® CHOP Trans-Reporting System
pFA-CHOP
pFC-MEK3
Uncharacterized pathway
c-Fos
pFA-cFos
Uncharacterized pathway
ATF2
pFA-ATF2
The PathDetect trans-reporting
systems include many choices of available pathways (Table
1). The original PathDetect systems incorporate a pathway-specific
trans-activator plasmid, an in-frame fusion of an activation domain
and the yeast GAL4 DNA binding domain (Figure
1A), and the pFR-Luc reporter plasmid (Figure
1B), which encodes firefly luciferase. While widely used because
it is sensitive, cost effective (does not require radioisotopes), and has
a broad linear range and minimal endogenous activity, luciferase does not
work in every research environment.
Fig.1A
Hence, we constructed four more reporter
plasmids with different encoded enzymes so PathDetect systems are
accessible to everyone studying the in vivo effects of a newly discovered
gene product or drug candidate. Expression of the vectors’ reporter
enzymes can be induced by any trans-activator with the GAL4
DNA-binding domain because they each carry a promoter that contains five
direct repeats of the 17-bp GAL4 binding element joined to the basic
promoter element (TATA box).
Fig. 1B
New Reporter Enzymes for
Trans-Reporter Plasmids
Assays for certain reporter enzymes
offer advantages for various laboratories, experiments, and
circumstances.2,3 Four new
reporter enzymes, ß-Gal, SEAP, CAT, and hrGFP, are now available in
PathDetect trans-reporter plasmids increasing the versatility of
this system.
The ß-gal gene functions well as a
reporter gene because its protein product is extremely stable and
resistant to proteolytic degradation in cellular lysates and, most
importantly, the enzyme is easily assayed. A spectrophotometer or a
microplate reader is the only instrument required. This reporter is
a good choice when a 96-well plate format is desired. Several assay
formats are available for b-gal activity
including colormetric, fluorescent, and chemiluminescent methods.
The greatest advantage of SEAP is that
the enzyme is secreted out of the cell after synthesis. Therefore, enzyme
activity can be continuously monitored without lysing the cells.
This also reduces background alkaline phosphatase activity caused by
cellular phosphatases and facilitates the automation of the sampling and
assay procedures.
The CAT enzyme has an in vivo half-life
of about 50 hours, a plus for when the desired result is one that compares
cumulative versus dynamic change. For this enzyme, a scintillation
counter is used.
Because expression of the hrGFP reporter
can be detected in vivo using a fluorescent microscope,
fluorescence-activated cell sorting, or a fluorometer—without disrupting
cells—this method is a quick and easy way to qualitatively assess
activation. The hrGFP reporter is particularly useful for high-throughput
drug discovery applications.
Quantitative Activation Readout with Three
Trans-Reporter Plasmids
To test the specificity of the
ß-gal, SEAP, and CAT reporter plasmids, we cotransfected a fusion
trans-activator plasmid and a known activator with each of the
trans-reporter plasmids into mammalian cells. The pFR-ßGal
trans-reporter plasmid and the pFA2-CREB trans-activator,
cotransfected into CHO cells, produced a 50-fold increase of ß-gal
activation in the presence of cAMP-activated protein kinase (PKA) (a known
CREB protein activator), compared with the negative control plasmid
pCMV-Script (Figure
2). The pFR-SEAP trans-reporter plasmid and
the trans-activator pFA2-CREB, also cotransfected into CHO cells,
showed that SEAP activity increased 14-fold in the presence of PKA,
compared with that in the sample cotransfected with the pCMV-Script
control plasmid (Figure
3). The pFR-CAT trans-reporter plasmid and the
pFA-cJun plasmid, cotransfected into NIH3T3 cells, demonstrated a 55-fold
increase in CAT expression in the presence of MEK kinase (MEKK) (a known
JNK activator), compared to cells cotransfected with the pCMV-Script
control plasmid.
Qualitative Activation In Vivo with One Reporter
Plasmid
The new PathDetect pFR-hrGFP
trans-reporter plasmid encodes the humanized Renilla green
fluorescent protein (hrGFP) derived from the sea pansy Renilla
reniformis. Stratagene’s hrGFP is unique among available GFP reporter
proteins because it does not possess the high cellular toxicity that may
distort the interpretation of experiments. Additionally, unlike other
reporter proteins, hrGFP activation is assessed without preparing cell
lysates. Instead, this reporter emits a high-intensity fluorescence when
activated that is easily detected in vivo by fluorescence microscopy or
fluorescence activated cell-sorting (FACS) analysis.
Fig. 5
We used the MAPK (Mitogen-Activated
Protein Kinase) and JNK signaling pathways to demonstrate activation of
the pFR-hrGFP trans-reporter plasmid. Since the MEK1 protein is a
known activator of the ELK1 protein, we used the pFC-MEK1 plasmid
(constitutively expresses this active kinase) to cotransfect the pFA2-ELK1
plasmid with the pFC-MEK1 plasmid and pFR-hrGFP plasmid and showed vivid
expression of the hrGFP trans-reporter protein (Figure 5). When cotransfected with GAL4-VP16, a well-known
trans-activator, the pFR-hrGFP reporter plasmid was also activated
. As expected, the control transfection of pFC-CMV,
pFR-hrGFP, and pFA2-ELK1 plasmids showed negligible hrGFP expression
.
Conclusions
Our four new PathDetect
trans-reporter plasmids offer more choices for measuring pathway
activation, making them available for use in a wider range of research
projects. For quantitative studies, choose the assay system with the
plasmid containing the encoded enzyme ß-gal, SEAP, or CAT. For quick, and
inexpensive qualitative studies, choose the plasmid containing the encoded
enzyme hrGFP—activation is easily detected in vivo without disrupting
cells. We tested the new trans-reporter plasmids in a variety of
cotransfection experiments and found that they attained the same high
level of activation readout as seen with the luciferase
trans-reporter plasmid that is incorporated in our original
PathDetect kit.
REFERENCES
1. Xu, L., Sanchez, T., and Zheng, C.-F. (1997)
Strategies 10: 1-3.
2. Bronstein, et al. (1994) Anal.
Biochem. 219: 169-181.
3. Kain, S. and Ganguly, S. (1995) In
Current Protocols in Molecular
Biology (Eds. F.M. Ausubel, et al.) John Wiley, New York.
* Patent Pending