Hedgehog Pathway

Hedgehog Pathway: The hedgehog pathway involves stem cell growth, many type tumor growth and CNS development. The pathway is equally important during vertebrate embryonic development. The pathway takes its name from its polypeptide ligand, an intercellular signaling molecule called Hedgehog. The mammalian hedgehog has three homologues, Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh).  The Hedgehog binds to their membrane receptor, activating the Gli proteins (transcription factors). The Gli proteins bind to Gli responsive element (Gli-RE) sequence in target’s promoter region, which leads to hedgehog signal’s target gene expression.

GenTarget developed a set of reporting lentivirus to measure the activation of Hedgehog signal pathway.  Those reporting lentivirus has a luminescent report (Luciferase, Renilla Luc) or a fluorescent report (GFP, RFP) or a  secreted SEAP report,  under a minimal CMV promoter (mCMV) that embedded with optimized tandem repeats of Gli responsive element sequence (5′-GACCACCCAC). When Gli proteins bind to Gli responsive element sequence, the downstream reporter is expressed as the result of activation for the minimal promoter. The reporter’s signal can be easily and rapidly readout via plate assays (luciferase assay or by fluorescent microscope).

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.

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 lentivector’s core expression cassette scheme below.

The premade, ready-to-use reporter lentivirus provides a much easier tool to monitor the activity of Hedgehog signaling pathways in virtually any mammalian cell type. It also allows to generate your own reporting cell line in your desired cell type for study or screen of pathway specific gene-knockdown, over-expression, or chemical / drug/protein treatment (such as Shh protein) in the cell based assay.

Click the Product Manual (pdf) for all available pathway reporter products.