CELL LINES
Contributor Information
- Name Tina Sket ; Andrii Domanskyi
- Institute University of Helsinki
Tool Details
- Tool name: XBP1 splicing reporter (XSARA) cell line
- Alternate names: XBP1-NLuc XBP1 splicing reporter cell line, XBP1-NLuc cells
- Tool type: Cell Lines
- Parental cell line: Flp-In HEK-293 T-Rex cells
- Organism: Human
- Tissue: Embryo
- Disease: cancer; diabetes; Chronic inflammatory syndromes; Neurodegenerative diseases
- Growth properties: Luciferase reporter
- Model: Reporter
- Conditional: Yes
- Conditional description: The expression of the reporter construct is induced by doxycycline (1 ÄÂÄ?g/ml final conc.) in the culture medium
- Description: 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 ParkinsonÄË?Â?Âs 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.
- Research area: Immunology; Cancer; Neurobiology
- Production details: Production details (How was the model produced): Flp-In HEK-293 T-Rex cells cultured in a Greiner CELLSTARÄÂÄË 10 cm dish were transfected with 1 ÄÂÄ?g of the target plasmid (pTO-sp-XBP1-NLuc-FRT, or pTO-sp-NLuc-FRT as a control) and 5 ÄÂÄ?g of plasmid pOG44 for the expression of Flp recombinase. The total amount of transfection mix for each plate was 500 ÄÂÄ?l. The cells were grown in the incubator for 48 h at 37ÄÂ?°C and 5% CO2, before the regular media was replaced with the selection media containing antibiotics (final concentrations of 15 ÄÂ??g/ml Blasticidin HCl and 100 ÄÂ??g/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
- For Research Use Only
Target Details
- Target: activation of IRE1 branch of unfolded protein response
Application Details
- Application notes: XBP1-NLuc reporter cell line provides a reliable reporter of IRE1 endonuclease activity, whose expression is increased during the ER stress, under various treatments and at different time points relative to treatments. Due to the effectiveness and accuracy, the XBP1-NLuc cell line can be further used in studying the regulation and activation of IRE1, as well as for the identification of ER-stress modulating molecules, which can be used for development of novel treatments for ER stress associated diseases, such as ParkinsonÄË?Â?Âs disease.
Handling
- Format: Frozen
- Growth medium: Dulbecco's Modified Eagle Medium (DMEM, pH 7.4; Gibco, #12491-015) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, Massachusetts, USA), NormocinÄË?Â? (100 ÄÂÄ?g/ml; N, InvivoGen, San Diego, USA), Blasticidin HCl (15 ÄÂ??g/ml; InvivoGen, Toulouse, France) and Hygromycin B (100 ÄÂ??g/ml; InvivoGen, Toulouse, France); re-plated 2-3 times per week in aseptic conditions following standard mammalian cell culture techniques.
- Shipping conditions: Dry ice
Documentation
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