CELL LINES
Contributor Information
- Name Tina Sket, Andrii Domanskyi
- Institute University of Helsinki, Institute of Biotechnology, HiLIFE
Tool Details
- Tool name: HEK293-NLuc (control) cell line
- Tool type: Cell Lines
- Tool sub-type: Continous
- Parental cell line: Flp-In HEK-293 T-Rex cell line (ThermoFisher Scientific, Waltham, MA)
- Organism: Human
- Tissue: Kidney
- Cancer type: Any cancer with endoplasmic reticulum (ER) stress and the activation of unfolded protein response (UPR) pathway
- Disease: Parkinson's disease; amyotrophic lateral sclerosis (ALS)
- Morphology: Epithelial
- Growth properties: Adherent
- Model: Knock-In
- Model description: Flp-In HEK-293 T-Rex cells (ThermoFisher Scientific, Waltham, MA) cultured in a Greiner CELLSTAR® 10 cm dish in DMEM supplemented with 10% FBS and 100 µg/ml Normocin were transfected with 1 ?g of the control plasmid (pTO-sp-NLuc-FRT) and 5 ?g of plasmid pOG44 for the expression of Flp recombinase. The total amount of transfection mix for each plate was 500 ?l. 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 µg/ml Blasticidin HCl and 100 µg/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, 10 colonies of control NLuc cell line were isolated.
- CRISPR: No
- Conditional: Yes
- Conditional description: Expression of NanoLuc luciferase is induced with 10 ng/ml doxycycline hyclate (Cat#D9891, Sigma-Aldrich, St. Louis, USA, DOX, in ethanol)
- Receptors of note: No
- Products or characteristics of interest: 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 HEK293-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 NanoLuc luciferase gene. The control HEK293-NLuc cells do not contain the XBP1 intron fragment in NanoLuc luciferase gene, and thus will express NanoLuc luciferase irresspectively of XBP1 splicing.
- Application: A control for XSARA assay
- 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 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 HEK293-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 NanoLuc luciferase gene. The control HEK293-NLuc cells do not contain the XBP1 intron fragment in NanoLuc luciferase gene, and thus will express NanoLuc luciferase irresspectively of XBP1 splicing.
- Research area: Cell Biology
- Production details: Flp-In HEK-293 T-Rex cells (ThermoFisher Scientific, Waltham, MA) cultured in a Greiner CELLSTAR® 10 cm dish in DMEM supplemented with 10% FBS and 100 µg/ml Normocin were transfected with 1 ?g of the control plasmid (pTO-sp-NLuc-FRT) and 5 ?g of plasmid pOG44 for the expression of Flp recombinase. The total amount of transfection mix for each plate was 500 ?l. 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 µg/ml Blasticidin HCl and 100 µg/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, 10 colonies of control NLuc cell line were isolated.
- For Research Use Only
Target Details
Application Details
- Application: A control for XSARA assay
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Application notes: Upon induction with doxycycline, HEK293-NLuc control cells will express NLuc protein irrespectively of XBP1 splicing, providing a control for XSARA assay.
Flp-In HEK-293 T-Rex cells (ThermoFisher Scientific, Waltham, MA) cultured in a Greiner CELLSTAR® 10 cm dish in DMEM supplemented with 10% FBS and 100 µg/ml Normocin were transfected with 1 ?g of the control plasmid (pTO-sp-NLuc-FRT) and 5 ?g of plasmid pOG44 for the expression of Flp recombinase. The total amount of transfection mix for each plate was 500 ?l. 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 µg/ml Blasticidin HCl and 100 µg/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, 10 colonies of control NLuc cell line were isolated.
For experiments, cells can be plated into Greiner CellStar 96-well clear-bottom plates at a density of approx. 10,000 cells/well.
Handling
- Format: Frozen
- Growth medium: DMEM (pH 7.4) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml Normocin (InvivoGen, San Diego, USA), 15 µg/ml Blasticidin HCl and 100 µg/ml Hygromycin B.
- Temperature: 37C
- Atmosphere: 5% CO2
- Storage medium: DMEM (pH 7.4) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml Normocin (InvivoGen, San Diego, USA), 15 µg/ml Blasticidin HCl and 100 µg/ml Hygromycin B + 5% sterile DMSO
- Storage conditions: Liquid Nitrogen; approx. 3x10^6 cells in a vial
- Shipping conditions: Dry ice
- Initial handling information: Add 10 mL pre-warmed cell culture media to 15 mL Falcon tube. Rapidly thaw frozen vial in water bath at 37°C for 30-60 sec. Pour cells from the vial to 15 mL Falcon tube (from Step 1). Spin down at 130x g at room temperature for 5 min. Carefully aspirate media. Carefully resuspend cell pellet in 10 ml fresh pre-warmed media. Plate on Petri dish or cell culture flask. Incubate at 37C in 5% CO2 incubator.
- Cultured in antibiotics?: Yes, 100 µg/ml Normocin (InvivoGen, San Diego, USA), 15 µg/ml Blasticidin HCl and 100 µg/ml Hygromycin B
- Mycoplasma free: Yes
- Biosafety level: 1
- Subculture routine: Replated at 1:5 dilution every 3-4 days
Documentation
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