KC-3319

293T-UAS-Luc2-GAL4-GRLBD-hRluc-Cell-Line

34314

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Background of 293T-UAS-Luc2-GAL4-GRLBD-hRluc-Cell-Line

GR is encoded by the gene NR3C1, which is located on chromosome 5q31-32 in humans, and acts as a ligand-inducible transcription factor belonging to the nuclear receptor superfamily. The molecular structure of GR includes four components: (1) an N-terminal transactivation domain (NTD), (2) a central DNA binding domain (DBD), (3) a C-terminal ligand-binding domain (LBD), and (4) a hinge region separating the DBD and the LBD. the LBD is a highly structured domain, consisting of 12 α-helices and four small β-strands, which forms a hydrophobic cavity for glucocorticoid binding and also contains an activation function (AF2). GR has also been shown to be intimately involved in the pathogenesis of many common cardiovascular diseases, including heart failure, atherosclerosis, sepsis, and myocardial ischemia. Dexamethasone is an agonist of GR, RU486 and cyproterone acetate are antagonists of GR, and progesterone and DHEA have antagonistic effects on GR. Gal4 is a ubiquitous transcriptional activator in eukaryotic cells. The sea pansy (Renilla reniformis) is another luciferase (RLuc) that has been extensively investigated; RLuc uses coelenterazine as a substrate and does not require ATP to produce light. With the use of commercially available molecular kits, the humanized forms of FLuc and RLuc (designated as hFLuc and hRLuc, respectively) were combined as dual reporters to image cultured cells in vivo because both enzymes used different substrates.

Specifications

Catalog Number	KC-3319
Cell Line Name	293T-UAS-Luc2-GAL4-GRLBD-hRluc-Cell-Line
Clone Number	3#
Host Cell Line	293T-UAS-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of GR 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+1µg/mL Puromycin+150µg/mL Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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
In Vivo Validation	NA

Cell Line Generation

293T-UAS-Luc2-GAL4-GRLBD-hRluc cell line was generated using lentivirus expressing exogenous hRluc, GAL4 and GRLBD gene sequence.

Characterization

Figure 1. 293T-UAS-Luc2-GAL4-GRLBD-hRluc cell line was seeded into the 96-well plate, and treated with dexamethasone at a maximum concentration of 100nM for 16 hours, then readout with Dual-Luciferase Reporter Assay System.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin 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 1:3-1:6 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

Chopra A. Gaussia princeps luciferase. 2008 Jan 31 [updated 2008 Feb 28]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004–2013. PMID: 20641352.
Elliott DA, Brand AH. The GAL4 system : a versatile system for the expression of genes. Methods Mol Biol. 2008;420:79-95. doi: 10.1007/978-1-59745-583-1_5. PMID: 18641942.
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.