KC-4569

CHOK1-MC2R-MRAP Cell Line

63923

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Background of CHOK1-MC2R-MRAP Cell Line

MC2R (Melanocortin 2 Receptor), also known as the ACTH receptor (ACTHR), is a G protein-coupled receptor primarily expressed in the adrenal cortex. It binds to adrenocorticotropic hormone (ACTH) to stimulate glucocorticoid synthesis. MRAP (Melanocortin 2 Receptor Accessory Protein) is a transmembrane protein essential for the proper trafficking of MC2R to the cell surface and enabling ACTH binding. Loss-of-function mutations in either gene cause Familial Glucocorticoid Deficiency (FGD). The MC2R/MRAP complex is a therapeutic target for ACTH-excess disorders like Cushing’s disease and congenital adrenal hyperplasia, with small molecule antagonists such as OMS1620 in development.

Specifications

Catalog Number	KC-4569
Cell Line Name	CHOK1-MC2R-MRAP Cell Line
NCBI/UniProt Accession Number	NM_000529.2, NM_178817.3
Clone Number	2#
Host Cell Line	CHOK1 cell line
Description	Stable CHOK1 cell line expressing exogenous human MC2R and MRAP gene
Quantity	One vial 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+10µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 26 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1-MC2R-MRAP cell line was generated using a lentiviral vector expressing the human MC2R and MRAP sequence.

Characterization

Figure 1: Characterization of human MC2R and MRAP overexpression in the CHOK1-MC2R-MRAP stable clone using qPCR.

Figure 2: Characterization of human MC2R and MRAP in the CHOK1-MC2R-MRAP stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Clark, Adrian John et al. “ACTH Antagonists.” Frontiers in endocrinology vol. 7 101. 5 Aug. 2016, doi:10.3389/fendo.2016.00101
2. Berruien, Nasrin N A, and Caroline L Smith. “Emerging roles of melanocortin receptor accessory proteins (MRAP and MRAP2) in physiology and pathophysiology.” Gene vol. 757 (2020): 144949. doi:10.1016/j.gene.2020.144949
3. Cooray, Sadani N et al. “Adrenocorticotropin resistance syndromes.” Endocrine development vol. 13 (2008): 99-116. doi:10.1159/000134828
4. Clark, Adrian J L, and Li Chan. “Stability and Turnover of the ACTH Receptor Complex.” Frontiers in endocrinology vol. 10 491. 26 Jul. 2019, doi:10.3389/fendo.2019.00491

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.