KC-5024

CHOK1-MC4R Cell Line

61154

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Background of CHOK1-MC4R Cell Line

Melanocortin 4 Receptor (MC4R) is a pivotal G protein-coupled receptor (GPCR) primarily expressed in the central nervous system, specifically within the paraventricular nucleus of the hypothalamus. It acts as a critical node in the leptin-melanocortin pathway, maintaining energy homeostasis by regulating appetite and energy expenditure. Mutations in the MC4R gene are the most common genetic cause of monogenic obesity and early-onset severe obesity. Current drug development centers on MC4R agonists. While therapies like Setmelanotide are approved for rare genetic obesity, research is rapidly shifting toward oral small-molecule agonists and combinations with GLP-1 drugs to treat broader obesity populations.

Specifications

Catalog Number	KC-5024
Cell Line Name	CHOK1-MC4R Cell Line
NCBI/UniProt Accession Number	NM_005912.2
Clone Number	3#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human MC4R 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
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

Cell Line Generation

CHOK1-MC4R Cell Line was generated using a lentiviral vector expressing the human MC4R sequence.

Characterization

Figure 1: Characterization of MC4R overexpression in the CHOK1-MC4R stable clone using qPCR.

Figure 2: Characterization of human MC4R in the CHOK1-MC4R stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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% RPMI1640 + 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. Wei, Ran et al. “MC4R in Central and Peripheral Systems.” Advanced biology vol. 7,9 (2023): e2300035. doi:10.1002/adbi.202300035
2. Liu, Zekun, and Victor J Hruby. “MC4R biased signalling and the conformational basis of biological function selections.” Journal of cellular and molecular medicine vol. 26,15 (2022): 4125-4136. doi:10.1111/jcmm.17441
3. Gonçalves, Juliana Pereira Lopes et al. “MC4R Agonists: Structural Overview on Antiobesity Therapeutics.” Trends in pharmacological sciences vol. 39,4 (2018): 402-423. doi:10.1016/j.tips.2018.01.004

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.