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	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% 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