KC-5767

A375-NRAS-Q61R-KI Cell Line

59932

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Background of A375-NRAS-Q61R-KI Cell Line

NRAS is a small GTPase belonging to RAS family, involved in the mitogen activated protein kinase (MAPK) signaling pathway, regulating cell growth, proliferation and differentiation in response to growth factors, cytokines and hormones. The role of RAS GTPase proteins is transmitting the signal from receptor tyrosine kinases (RTKs), at the cell surface, to transcription factors and cell cycle proteins in the nucleus. Activated RAS proteins stimulate not only the MAPK signaling pathway, but also the phosphoinositide 3-kinase (PI3K)/AKT pathway, which contributes to melanoma development through effects on the metabolism and other factors. Approximately 80% of NRAS mutations in melanoma occur at codon 61 (exon 3), involving amino acid changes from glutamine (Q) to arginine (R), lysine (K) or leucine (L), and locking the NRAS protein into a GTP-bound state, thereby impairing its GTPase activity. The remaining 20% of melanoma NRAS mutations fall in codons 12 and 13 (exon 2), leading to an amino acid substitution from glycine (G), to aspartic acid (D), which inhibits binding of GTPase activating protein to NRAS. According to Murphy et al., NRAS codon 61 mutations (p.Q61R, p.Q61K, and p.Q61L) empower the activation of MAPK signaling pathway more than the NRAS mutations at codons 12 and 13.

Specifications

Catalog Number	KC-5767
Cell Line Name	A375-NRAS-Q61R-KI Cell Line
Clone Number	2C1
Host Cell Line	A375
Description	Stable A375 clone expressing exogenous NRAS gene bearing Q61R mutations
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split saturated culture 1:4-1:5 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 28 hours
Mycoplasma Status	Negative

Cell Line Generation

A375-NRAS-Q61R-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A375-NRAS-Q61R-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of A375-NRAS-Q61R-KI cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of dose-response curves for NRAS inhibitors on A375 and A375-NRAS-Q61R-KI cells.

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 saturated culture 1:4-1:5 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

Zupa A, Vita G, Omer LC, Calice G, Conca R, Improta G. NRASQ61K/R/L Mutant Allele Frequency in Melanoma and its Correlation With Clinicopathological Characteristics. Anticancer Res. 2025 Mar;45(3):1015-1024. doi: 10.21873/anticanres.17488. PMID: 40037888.