Notch Signal Pathway

Notch Signal Pathway: The Notch signaling pathway is a highly conserved cell signaling system present in most multi-cellular organisms. Mammals possess four different notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The notch receptor is a single-pass transmembrane receptor protein.

Notch signal pathway is normally triggered via direct cell-to-cell contact The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain transmembrane receptor (NOTCH1 / NOTCH2 /NOTCH3 /NOTCH4) on a neighboring cell, induce proteolytic cleavage and release of the intracellular domain (NICD), which enters the cell nucleus, and form a complex with trasncription factors (CBF1 / RBP-JK) to activate the Notch-responsive gene expression.

The Notch binding allows groups of cells to organize themselves, such that, if one cell expresses a given trait, this may be switched off in neighbouring cells by the intercellular notch signal. In this way, groups of cells influence one another to make large structures. Thus, lateral inhibition mechanisms are keys to Notch signaling. Lin-12 and Notch mediate binary cell fate decisions, and lateral inhibition involves feedback mechanisms to amplify initial differences. The Notch signaling pathway is important for cell-cell communication, which involves gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life.

GenTarget developed a set of reporting lentivirus to monitor Notch signaling 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 RBP-JK  transcriptional responsive element (RBP-JK TRE) sequence motif (5′- CGTGGGAA). When transcription factors bind to RBP-JK-TRE, the downstream reporter is expressed as the result of activation for the minimal promoter. This pathway lentivirus can be verified by the treatment of Notch1 protein.

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 lentivector core scheme below.

The premade, ready-to-use reporter lentivirus provides a much easier tool to monitor the activity of Notch 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 in the cell based assay.

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