KC-5519

CT26-KRAS-V8M-G12A-G13D-KI Cell Line

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Background of CT26-KRAS-V8M-G12A-G13D-KI Cell Line

Kirsten rat sarcoma viral oncogene homologue (KRAS) is the best-known oncogene with the highest mutation rate among all cancers and is associated with a series of highly fatal cancers, including pancreatic ductal adenocarcinoma (PDAC), nonsmall-cell lung cancer (NSCLC), and colorectal cancer (CRC). The identification of tumor driver genes and the development of specific inhibitors have revolutionized cancer treatment strategies and clinical outcomes.

Specifications

Catalog Number	KC-5519
Cell Line Name	CT26-KRAS-V8M-G12A-G13D-KI Cell Line
Clone Number	3A4
Host Cell Line	CT26
Description	Stable CT26 clone expressing exogenous KRAS gene bearing G12A-G13D mutations, No.3A4
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
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 1-2 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

CT26-KRAS-G12A-G13D-KI cell line was generated using the CRISPR method

Characterization

Figure 1: Characterization of CT26-KRAS-G12A-G13D-KI-3A4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of CT26-KRAS-G12A-G13D-KI-3A4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of dose-response curves for KRAS inhibitors on CT26 and CT26-KRAS-G12A-G13D-KI-3A4 cells.

Figure 4: Characterization of CT26-KRAS-G12A-G13D-KI-3A4 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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 1-2 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Lamei Huang, Zhixing Guo, Fang Wang & Liwu Fu. KRAS mutation: from undruggable to druggable in cancer. Signal Transduction and Targeted Therapy (2021) 6:386 ; https://doi.org/10.1038/s41392-021-00780-4.