#219001 (pFR-CAT Plasmid) BioVector NTCC质粒载体菌种细胞基因保藏中心

#219001 (pFR-CAT Plasmid)

PathDetect Control Plasmid Reporting Systems RUO
The PathDetect trans-reporting systems allows you to determine the direct or indirect involvement of new gene products, growth factors, and drug candidates in specific pathways. They are available as plasmid vectors with a luciferase reporter for cotransfection with the gene of interest.


Investigate whether your protein or compound influences the activation of any of the six transcription factors offered. For ultimate flexibility, the pFA-CMV vector includes a multiple cloning site for inserting and expressing any transcription factor of interest.

#219000 (PathDetect c-Jun trans-Reporting System)
#219005 (PathDetect Elk1 trans-Reporting System)
#219010 (PathDetect CREB trans-Reporting System)
#219015 (PathDetect CHOP trans-Reporting System)
#219026 (pFA-ATF2 Plasmid)
#219031 (pFA-cFos Plasmid)
#219036 (pFA-CMV Plasmid)
#219001 (pFR-CAT Plasmid)
#219002 (pFR-βGal Plasmid)
#219004 (pFR-SEAP Plasmid)

The PathDetect in vivo signal transduction pathway trans-reporting systems
are designed for specific, rapid, and convenient assessment of the in vivo
activation of transcription activators and upstream signal transduction
pathways.1, 2 The systems are useful for identifying whether a newly cloned
gene is involved in a signal transduction pathway, and if so, at which step of
the pathway. These reporting systems can also be used to study the in vivo
effects of growth factors, drug candidates, and extracellular stimuli on
PathDetect signal transduction pathways. Some of the reporters may also be
used for cloning novel signal transduction genes, and may be useful for
identifying drug candidates in a high-throughput format.
Each PathDetect trans-reporting system includes a unique fusion transactivator plasmid that expresses a fusion protein (see Figure 2 in
Appendix I). The fusion protein is a trans-acting, pathway-specific
transcriptional activator (i.e., a fusion trans-activator protein). The fusion
trans-activator protein consists of the activation domain of either the
c-Jun,3,–6 Elk1,6–9 CREB,6, 10 CHOP,2, 11 ATF2,12, 13 or c-Fos12, 13 transcriptional
activator fused with the yeast GAL4 DNA binding domain (DBD),14, 15
residues 1–147. The transcriptional activators c-Jun, Elk1, CREB, and
CHOP are phosphorylated and activated by c-Jun N-terminal kinase (JNK),
mitogen-activated protein kinase (MAPK), cyclic AMP-dependent kinase
(PKA), or p38 MAP kinase, respectively, and their activity reflects the in
vivo activation of these kinases and the corresponding signal transduction
pathways. The signal transduction pathways leading to the phosphorylation
of the transcription activators ATF2 and c-Fos are not characterized.
The fusion trans-activator plasmid contains the human cytomegalovirus
(CMV) immediate early promoter to drive the constitutive expression of the
trans-activator protein in a wide variety of eukaryotic cell lines. Selection in
bacteria is made possible by the kanamycin-resistance gene, which is under
control of the prokaryotic β-lactamase promoter. The neomycin-resistance
gene, driven by the SV40 early promoter, provides stable selection with
G418 in mammalian cells.
The pFA-CMV trans-Activator Plasmid
The pFA-CMV plasmid is a mammalian expression vector that is designed
to allow the study of any transcription activator and signal transduction
pathway of interest. The pFA-CMV plasmid is designed for convenient
insertion of the activation domain sequence of any transcription activator.
The fusion trans-activator protein expressed by the pFA-CMV plasmid
consists of the activation domain of the transcription activator of interest
fused with the DNA binding domain of the yeast GAL4.

The pFR-Luc Reporter Plasmid
The pFR-Luc reporter plasmid contains a synthetic promoter with five
tandem repeats of the yeast GAL4 binding sites that control expression of
the Photinus pyralis (American firefly) luciferase gene (see Figure 3 in
Appendix I). The DNA binding domain of the fusion trans-activator protein
binds to the reporter plasmid at the GAL4 binding sites. When a fusion
trans-activator plasmid, reporter plasmid, and an uncharacterized gene are
cotransfected into mammalian cells, direct or indirect phosphorylation of the
transcription activation domain of the fusion trans-activator protein by the
uncharacterized gene product will activate transcription of the luciferase
gene from the reporter plasmid (Figure 1). Expression (or activity) levels of
luciferase reflect the activation status of the signaling events.
Alternate Reporter Proteins
We have constructed a series of plasmids that enable the use of the
PathDetect system with reporter proteins other than luciferase. These
alternate reporter proteins may offer advantages for certain laboratories or
experiments. These reporter proteins include chloramphenicol
acetyltransferase (CAT), β-galactosidase, and the secreted alkaline
phosphatase (SEAP). The plasmids have the same backbone as the pFR-Luc
plasmid and have been validated to function with the PathDetect reporting
systems (see Figure 3 in Appendix I). For protocols outlining the use of
these alternate reporter proteins see Alternate Reporter Plasmids.

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