Hypoxia Pathway
Hypoxia Pathway: Hypoxia-inducible factors (HIFs) are transcription factors that respond to the decreases in oxygen (hypoxia) in the cellular environment. Low oxygen conditions activate the hypoxia signaling pathway. The hypoxia-inducible factor-1 (HIF1) protein is a key regulator of oxygen homeostasis. HIFs are hydroxylated and degradation at the presence of oxygen. Under low oxygen conditions, the hydroxylase is inhibited which allows HIF to accumulate. The HIF binds to hypoxia transcriptional response element (H-RE) in the promoter region of the target genes, initiating gene expression.
HIF1 is known to induce transcription of more than 60 genes that involve in biological processes such as angiogenesis, erythropoiesis vasculogenesis, wound healing, cell proliferation and survival, as well as in cancer progression, cardiovascular disease, tumor angiogenesis and chronic inflammation.
GenTarget developed a set of reporting lentivirus to monitor the activity of HIF-regulated signal transduction pathways. 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 hypoxia transcriptional response element (H-RE) sequence motif (5′- GACGTGCG, TACGTGGG, TACGTGCT). When HIFs binds to hypoxia transcriptional response element (H-RE), it h induces the downstream reporter’s expression as the result of activation for the minimal promoter. This pathway lentivirus can be verified (stimulated) by CoCl2.
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 Hypoxia 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.