KC-2131

293T-cyno-EP4-Cell-Line

23452

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Background of 293T-cyno-EP4-Cell-Line

PTGER4 (Prostaglandin E Receptor 4), also known as EP4. The EP4 prostanoid receptor is one of four receptor subtypes for prostaglandin E2, it belongs to the family of G protein-coupled receptors, it is a Protein Coding gene. The protein encoded by this gene is a member of the G-protein coupled receptor family. This receptor can activate T-cell factor signaling. It has been shown to mediate PGE2 induced expression of early growth response 1 (EGR1), regulate the level and stability of cyclooxygenase-2 mRNA, and lead to the phosphorylation of glycogen synthase kinase-3. Diseases associated with PTGER4 include Skin Nasal Cell Carcinoma With Sebaceous Differentiation and Scrotum Paget's Disease.

Specifications

Catalog Number	KC-2131
Cell Line Name	293T-cyno-EP4-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno EP4 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
In Vivo Validation	NA

Cell Line Generation

293T cyno-EP4 cell line was generated using a lentiviral vector expressing the cyno-EP4 sequence.

Characterization

Figure 1: Characterization of endogenous cyno EP4 expression in 293T cells using QPCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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

Jiang W, Jin Y, Zhang S, Ding Y, Huo K, Yang J, Zhao L, Nian B, Zhong TP, Lu W, Zhang H, Cao X, Shah KM, Wang N, Liu M, Luo J. PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis. Bone Res. 2022 Mar 9;10(1):27. doi: 10.1038/s41413-022-00201-4. PMID: 35260562; PMCID: PMC8904489.
Yokoyama U, Iwatsubo K, Umemura M, Fujita T, Ishikawa Y. The prostanoid EP4 receptor and its signaling pathway. Pharmacol Rev. 2013 Jun 17;65(3):1010-52. doi: 10.1124/pr.112.007195. PMID: 23776144.
Majumder M, Nandi P, Omar A, Ugwuagbo KC, Lala PK. EP4 as a Therapeutic Target for Aggressive Human Breast Cancer. Int J Mol Sci. 2018 Mar 29;19(4):1019. doi: 10.3390/ijms19041019. PMID: 29596308; PMCID: PMC5979567.