KC-4359

293T-GPR75-KO-2C4-Cell-Line

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Background of 293T-GPR75-KO-2C4-Cell-Line

G-protein coupled receptors (GPCRs) are important membrane molecules involved in regulating physiological responses to hormones, neurotransmitters, and various environmental stimuli.G protein-coupled receptor 75 (GPR75), a member of the GPCR family, it was first reported by Tarttelin et al. in 1999. It is a protein composed of 540 amino acids located on human chromosome 2p16, containing two exons. The first exon includes an untranslated sequence, while the second exon contains the complete coding region of GPR75, with no similarity to other known genes or transcripts. GPR75 has typical structural features of GPCRs, including seven transmembrane domains, N-terminal glycosylation sites, and a C-terminal with multiple serine and threonine phosphorylation sites, but it has low structural homology with other similar receptors. GPR75 is widely expressed in the liver, vascular endothelium, eyes, kidneys, and adipose tissue, playing an important role in the signaling mechanisms of metabolic syndrome.

Specifications

Catalog Number	KC-4359
Cell Line Name	293T-GPR75-KO-2C4-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T cell line with human GPR75 gene knockout, No.2C4
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
Selection Marker	N/A
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:4~1:5 every 2~3 days; seed out at about 1-3 x 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-GPR75-KO 2C4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-GPR75-KO 2C4 Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-GPR75-KO 2C4 Cell Line stable clone using RT-PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS)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 the saturated culture at a ratio of 1:4 ~ 1:5 every 2~3 days; seed out at about 1-3 x 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

Wang S, Gao S, Wang F. Effect and mechanism of GPR75 in metabolic dysfunction-related steatosis liver disease. Int J Med Sci. 2024 Sep 9;21(12):2343-2347. doi: 10.7150/ijms.101094. PMID: 39310267; PMCID: PMC11413904.