CREB (cAMP-PKA) Pathway
About CREB (cAMP-PKA) Pathway:
cAMP-PKA Signal Pathway: Many extracellular and intracellular signaling pathways involve the cAMP response element (CRE) which has a general motif of 5′-TGACGTCA-3’. Many pathway signals, like cAMP, GPCR, and others, activate the CREB (cAMP Response Element Binding proteins) which in turn binds to the cAMP response motif, resulted in the transcription of the downstream gene / reporter.
The PKA enzyme is also known as cAMP-dependent enzyme because it gets activated only if cAMP is present. In cAMP/PKA pathway, PKA activate CREB which then binds to CRE motif, and induces the downstream gene expression. This pathway is critical to numerous life processes and living organisms. The CRE motif (as Transcription response element, TRE) provides a system for study of cAMP/PKA pathway’s activation or inhibition.
Detect CREB (cAMP-PKA) Pathway:
GenTarget developed a set of reporting lentivirus for monitoring or manipulating the CRE pathway’s activity in any of your desired cell types. Those reporting lentivirus has a luminescent report (Luciferase, Renilla Luc) or a fluorescent report (GFP, RFP) or a secreted SEAP report, under a minimal promoter that embedded with optimized tandem repeats of the CRE motif (5’-TGACGTCA-3’). When cellular cAMP level is elevated, the activated CREB will binds to the minimal promoter’s TRE motif, which increases the expression of the downstream luminescent or fluorescent report.
The luciferase signal can be measured via Luciferase assay. The fluorescent reporter can be detected via its fluorescent signal. The SEAP secreted into cell culture supernatant, which allows to determine reporter activity without disturbing the cells, does not require the preparation of cell lysates and can be used for kinetic studies.
How to Use Report Pathway Lentivirus:
Those reporting lentivirus also contains a constitutively expressed fluorescent selection marker or an antibiotic selection marker under the RSV promoter, which makes it easier to select the stably infected signal reporting cells (to generate pathway specific sensor cell lines), or provides internal reference for virus transduction efficiency when a fluorescent marker is under the RSV promoter. See the scheme below for lentivector’s core expression cassette.
The premade, ready-to-use reporter lentivirus provides a much easier tool to monitor the activity of cAMP/PKA signaling pathways in virtually any mammalian cell type. It also allow to generate your own reporting cell line in your desired cell type for study or screen of pathway specific compounds or drugs (such as Forskolin), in the cell based assay.
Click the Product Manual (pdf) for all available product for this pathway reporter.