KC-6268

293T-GRE-Luc2-MR-Cell-Line

61187

Home » 293T-GRE-Luc2-MR-Cell-Line

Background of 293T-GRE-Luc2-MR-Cell-Line

Glucocorticoids (GCs) and their analogs regulate downstream gene expression through the glucocorticoid receptor (GR, also known as NR3C1), which is a member of the nuclear receptor superfamily of ligand-activated transcription factors. GCs diffuse into the cytoplasm, bind to GR, and are then translocated to the nucleus, where they bind to glucocorticoid response elements (GREs) in the promoters of target genes, thereby activating or repressing gene expression. GCs also modulate gene expression via non-GR pathways, such as through cAMP response element-binding (CREB) protein, activator protein (AP)-1, and NF-κB. These receptors have several major domains, including an amino-terminal activation domain, a central zinc-finger DNA-binding domain, and a carboxy-terminal ligand-binding domain. This system can be used to study the activation levels of glucocorticoid-mediated signaling pathways, drug research, and gene overexpression and RNAi phenotypic analysis.The mineralocorticoid receptor is a steroid hormone receptor that is well known for its involvement in fluid and electrolyte homeostasis in epithelial cells present in the distal nephron. The inappropriate activation of this receptor is now known to be implicated in various pathophysiological mechanisms in heart failure.

Specifications

Catalog Number	KC-6268
Cell Line Name	293T-GRE-Luc2-MR-Cell-Line
Clone Number	2A2
Host Cell Line	293T-GRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of MR signaling pathway.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS+150µg/mL Hygromycin B+1µg/mL Puromycin
Selection Marker	Puromycin \| Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-GRE-Luc2-MR Cell Line was generated using a lentiviral vector expressing MR sequence.

Characterization

Figure 1. 293T-GRE-Luc2-MR cell line was seed into the 96-well plate, and treated with Aldosterone at a maximum concentration 1.56 ng/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Figure 2. 293T-GRE-Luc2-MR cell line was seed into the 96-well plate, and treated with Finerenone at a maximum concentration 10 µM diluted 3.16-fold for 1 hour, and then Aldosterone treatment in the concentrations of 1 ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, 150µg/mL Hygromycin B and 1µg/mL Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Liu B, Zhang TN, Knight JK, Goodwin JE. The Glucocorticoid Receptor in Cardiovascular Health and Disease. Cells. 2019 Oct 9;8(10):1227. doi: 10.3390/cells8101227. PMID: 31601045; PMCID: PMC6829609.
2. Wu T, Shao Y, Li X, Wu T, Yu L, Liang J, Zhang Y, Wang J, Sun T, Zhu Y, Chang X, Wang S, Chen F, Han X. NR3C1/Glucocorticoid receptor activation promotes pancreatic β-cell autophagy overload in response to glucolipotoxicity. Autophagy. 2023 Sep;19(9):2538-2557. doi: 10.1080/15548627.2023.2200625. Epub 2023 Apr 20. PMID: 37039556; PMCID: PMC10392762.
3. Karthigan N, Lockwood S, White A, Yang J, Young MJ. Mineralocorticoid receptor antagonists, heart failure and predictive biomarkers. J Endocrinol. 2022 Apr 15;253(3):R65-R76. doi: 10.1530/JOE-21-0323. PMID: 35266453.
4. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987 Jul 17;237(4812):268-75. doi: 10.1126/science.3037703. PMID: 3037703.