KC-4932

293T-CRE-Luc2-TSHR-Cell-Line

60431

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Background of 293T-CRE-Luc2-TSHR-Cell-Line

The protein encoded by TSHR is a membrane protein and a major controller of thyroid cell metabolism. The encoded protein is a receptor for thyrothropin and thyrostimulin, and its activity is mediated by adenylate cyclase. The phenotype spectrum is wide: from severe congenital hypothyroidism to mild hyperthyrotropinemia. Over 250 TSHR variants have been published, many uncharacterized in vitro. TSHR is sulfated on tyrosines in a motif located downstream of the C-terminal cysteine cluster. Sulfation of one of the two tyrosines in the motif is mandatory for high-affinity binding of TSH and activation of the receptor. Site-directed mutagenesis experiments indicate that the motif, which is conserved in all members of the glycoprotein hormone receptor family, seems to play a similar role in the LH/CG and FSH receptors.

Specifications

Catalog Number	KC-4932
Cell Line Name	293T-CRE-Luc2-TSHR-Cell-Line
Clone Number	1#
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of TSHR signaling pathway.
Quantity	One vial 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	70% DMEM+10% FBS+1µg/mL Puromycin
Selection Marker	Puromycin
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-CRE-Luc2-TSHR Cell Line was generated using a lentiviral vector expressing TSHR sequence.

Characterization

Figure 1. 293T-CRE-Luc2-TSHR cell line was seed into the 96-well plate, and treated with TSH at a maximum concentration 1000 ng/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Figure 2. Characterization of 293T-CRE-Luc2-TSHR using FACS.

Figure 3.Characterization of 293T-CRE-Luc2-TSHR using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, 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. Yeste D, Baz-Redón N, Antolín M, Garcia-Arumí E, Mogas E, Campos-Martorell A, González-Llorens N, Aguilar-Riera C, Soler-Colomer L, Clemente M, Fernández-Cancio M, Camats-Tarruella N. Genetic and Functional Studies of Patients with Thyroid Dyshormonogenesis and Defects in the TSH Receptor (TSHR). Int J Mol Sci. 2024 Sep 18;25(18):10032. doi: 10.3390/ijms251810032. PMID: 39337518; PMCID: PMC11432690.
2. Costagliola S, Panneels V, Bonomi M, Koch J, Many MC, Smits G, Vassart G. Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors. EMBO J. 2002 Feb 15;21(4):504-13. doi: 10.1093/emboj/21.4.504. PMID: 11847099; PMCID: PMC125869.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.