KC-5918

CHOK1-CRE-Luc2-TSHR-Mc4-Cell-Line

59295

Home » CHOK1-CRE-Luc2-TSHR-Mc4-Cell-Line

Background of CHOK1-CRE-Luc2-TSHR-Mc4-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-5918
Cell Line Name	CHOK1-CRE-Luc2-TSHR-Mc4-Cell-Line
Clone Number	2A3
Host Cell Line	CHOK1-CRE-Luc2
Description	Stable CHOK1 cell line expressing exogenous luciferase gene under the control of TSHR 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+5µg/mL Puromycin+375µg/mL Hygromycin B
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

CHOK1-CRE-Luc2-TSHR-Mc4 Cell Line was generated using a lentiviral vector expressing TSHR sequence.

Characterization

Figure 1. CHOK1-CRE-Luc2-TSHR-Mc4 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. CHOK1-CRE-Luc2-TSHR-Mc4 cell line was seed into the 96-well plate, and treated with M22-hIgG1 at a maximum concentration 20µg/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Figure 3. CHOK1-CRE-Luc2-TSHR-Mc4 cell line was seed into the 96-well plate, and treated with K170-hIgG1 and IgG1 at a maximum concentration 20µg/mL diluted 3.16-fold for 1 hour, and then TSH treatment in the concentrations of 10000 ng/mL for 16 hours, then readout with Bright-Glo system.

Figure 4. CHOK1-CRE-Luc2-TSHR-Mc4 cell line was seed into the 96-well plate, and treated with K170-hIgG1 and IgG1 at a maximum concentration 20µg/mL diluted 3.16-fold for 1 hour, and then M22-hIgG1 treatment in the concentration of 2 µg/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 375µg/mL Hygromycin and 5µg/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% RPMI1640 + 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.