Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which leads to the activation of unfolded protein response (UPR) through three transmembrane protein sensors located in the ER membrane. The sensors correspond to three branches of the UPR, namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) branches. Upon ER stress, IRE1 dimerizes and oligomerizes, and its endonuclease domain is activated. It specifically targets X-box-binding protein 1 (XBP1) mRNA, from which a 26 nt intron is spliced. This allows a complete translation of spliced XBP1 mRNA into a functional protein that acts as a transcription factor. Together with the other pathways, the UPR leads to a decrease in the protein folding load by causing a reduction in the general level of protein translation, and by inducing the expression of protein folding machinery. However, if the UPR is activated continuously for a long time, the apoptotic pathway will be triggered, and the cell will die.ER stress and UPR are associated with various disorders, such as some types of cancer, diabetes, chronic inflammatory syndromes, and particularly neurodegeneration. For example, in Parkinson’s disease, it was suggested that prolonged ER stress induces the extensive apoptosis of dopaminergic neurons in substantia nigra pars compacta region of the midbrain.The XBP1-NLuc cells are used to identify compounds affecting IRE1 branch of the UPR. The reporter is correctly spliced by activated IRE1, due to the presence of the XBP1 intron fragment in Nano luciferase gene.
The expression of the reporter construct is induced by doxycycline (1 ug/ml final conc.) in the culture medium
Production details:
How was the model produced: Flp-In HEK-293 T-Rex cells cultured in a Greiner CELLSTAR 10 cm dish were transfected with 1 ug of the target plasmid (pTO-sp-XBP1-NLuc-FRT, or pTO-sp-NLuc-FRT as a control) and 5 ug of plasmid pOG44 for the expression of Flp recombinase. The total amount of transfection mix for each plate was 500 ul. The cells were grown in the incubator for 48 h at 37°C and 5% CO2, before the regular media was replaced with the selection media containing antibiotics (final concentrations of 15 ug/ml Blasticidin HCl and 100 ug/ml Hygromycin B). The selection media was changed every 2-4 days, until the untransfected cells have died and the successfully transfected cells formed visible colonies after approximately 2-3 weeks. The colonies were transferred to a Greiner CellStar 6-well, clear-bottom cell culture plate into separate wells. In total, 12 colonies of XBP1-NLuc cell line and 10 colonies of control NLuc cell line were isolated
Parental cell line:
Flp-In HEK-293 T-Rex cells
Disease:
Cancer
Cat. #:
160833
Unit size:
1×10^6 cells / vial
Research Fields:
Cancer; Immunology; Neurobiology
Organism:
Human
Tissue:
Embryo
Model:
Reporter
Growth properties:
Luciferase reporter
Format:
Frozen
Shipping conditions:
Dry ice
Growth medium:
Dulbecco’s Modified Eagle Medium (DMEM, pH 7.4; Gibco) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), Normocin (100 ug/ml; InvivoGen), Blasticidin HCl (15 ug/ml; InvivoGen) and Hygromycin B (100 ug/ml; InvivoGen); re-plated 2-3 times per week in aseptic conditions following standard mammalian cell culture techniques. The expression of the reporter construct is induced by doxycycline (1 ug/ml final conc.) in the culture medium.
Recommended controls:
NLuc cells, expressing luciferase reporter devoid of XBP1 intron
Human buccal mucosa modeling; Permeability, absorption and metabolism studies of various substances and enzymatically labile drugs; Drug delivery studies
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