KC-4704

293T-mouse-TSHR Cell Line

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Background of 293T-mouse-TSHR Cell Line

The protein encoded by this gene 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. Defects in this gene are a cause of several types of hyperthyroidism. Three transcript variants encoding different isoforms have been found for this gene.

Specifications

Catalog Number	KC-4704
Cell Line Name	293T-mouse-TSHR Cell Line
Clone Number	4#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous mouse-TSHR gene
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
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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-mouse-TSHR cell line was generated using lentivirus expressing mouse-TSHR sequence.

Characterization

Figure 1. Characterization of mouse-TSHR over-expression in the 293T-mouse-TSHR stable clone using qPCR.

Figure 2. Characterization of mouse-TSHR over-expression in the 293T-mouse-TSHR stable clone using PCR.

Cell Resuscitation

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

Cell Freezing

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

References

Neumann S, Geras-Raaka E, Marcus-Samuels B, Gershengorn MC. Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization. FASEB J. 2010 Oct;24(10):3992-9. doi: 10.1096/fj.10-161745. Epub 2010 Jun 10. PMID: 20538910; PMCID: PMC2996905.
Dai YL, Cai DH, Chen H, Zhang Y, Li J. [Transcription and promoter hypermethylation of thyroid stimulating hormone receptor gene in human papillary thyroid carcinoma]. Nan Fang Yi Ke Da Xue Xue Bao. 2010 Jan;30(1):114-7. Chinese. PMID: 20117998.