KC-3271

293T-PTH1R Cell Line

26479

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Background of 293T-PTH1R Cell Line

PTH1R, also known as PTHR1, is a protein that belongs to the G-protein coupled receptor 2 family. PTH1R is a receptor for parathyroid hormone and for parathyroid hormone-related peptide. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase, and phosphatidylinositol-calcium second messenger system. Diseases associated with PTH1R include Jansen type metaphyseal chondrodysplasia and Blomstrand type Chondrodysplasia. Among its related pathways are endochondral ossification with skeletal dysplasias and GPCR downstream signaling.

Specifications

Catalog Number	KC-3271
Cell Line Name	293T-PTH1R Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous human PTH1R 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 1:4-1:5 every 2-3 days; seed out at about 1-2 × 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 human PTH1R Cell Line was generated using a lentiviral vector expressing the human PTH1R sequence.

Characterization

Figure 1: Characterization of human PTH1R overexpression in the 293T human PTH1R stable clone using PCR sequence

Figure 2: Characterization of human PTH1R overexpression in the 293T human PTH1R stable clone using FACS.

Figure3: Characterization of endogenous human PTH1R expression in 293T using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1-2 × 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. Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2. PMID: 18611381 PMCID: PMC2601695
2. Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. PMID: 20172855 PMCID: PMC2852981
3. Yamaguchi T, Hosomichi K, Narita A, Shirota T, Tomoyasu Y, Maki K, Inoue I (Jul 2011). Exome resequencing combined with linkage analysis identifies novel PTH1R variants in primary failure of tooth eruption in Japanese. Journal of Bone and Mineral Research. 26 (7): 1655–61.