KC-3476

293T-CRE-Luc2-PTH1R Cell Line

26447

Home » 293T-CRE-Luc2-PTH1R Cell Line

Background of 293T-CRE-Luc2-PTH1R Cell Line

PTH1R is a member of the G-protein coupled receptor family 2. It is a receptor for parathyroid hormone (PTH) and for parathyroid hormone-like hormone (PTHLH). The activity of this receptor is mediated by G proteins which activate adenylyl cyclase and also a phosphatidylinositol-calcium second messenger system. Defects in this receptor are known to be the cause of Jansen's metaphyseal chondrodysplasia (JMC), chondrodysplasia Blomstrand type (BOCD), as well as enchodromatosis.

Specifications

Catalog Number	KC-3476
Cell Line Name	293T-CRE-Luc2-PTH1R Cell Line
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of PTH1R 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 1:4-1:8 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
In Vivo Validation	NA

Cell Line Generation

293T cell line was generated using lentivirus expressing PTH1R sequence.

Characterization

Figure 1. 293T-CRE-Luc2-PTH1R and 293T-CRE-Luc2 cell line were seeded into the 96-well plate, and treated with PTH(1-34) at a maximum concentration of 100μg/mL for 16 hours, then readout with Bright-Glo 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 1:4-1:8 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.Pioszak AA, Harikumar KG, Parker NR, Miller LJ, Xu HE. Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. J Biol Chem. 2010 Apr 16;285(16):12435-44. doi: 10.1074/jbc.M109.093138. Epub 2010 Feb 19. PMID: 20172855; PMCID: PMC2852981.
2.Shimomura-Kuroki J, Farooq M, Sekimoto T, Amizuka N, Shimomura Y. Characterization of a PTH1R missense mutation responsible for Jansen type metaphyseal chondrodysplasia. Odontology. 2017 Apr;105(2):150-154. doi: 10.1007/s10266-016-0247-4. Epub 2016 May 9. PMID: 27160269.