KC-3985

AGS-KRAS-G12D-Y96D-KI Cell Line

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Background of AGS-KRAS-G12D-Y96D-KI Cell Line

The KRAS gene, encoding the Kirsten rat sarcoma viral oncogene homolog, is a member of the RAS family of small GTPases. KRAS plays a crucial role in transmitting signals from cell surface receptors to downstream effectors, primarily through the MAPK/ERK and PI3K/AKT signaling pathways. These pathways regulate various cellular processes, including proliferation, differentiation, survival, and apoptosis.Recent advances in understanding the molecular mechanisms of KRAS signaling and the development of targeted therapies have opened new avenues for treating KRAS-driven cancers. Small molecule inhibitors that directly target mutant KRAS proteins, such as AMG 510 and MRTX849, have shown promising results in clinical trials, offering hope for patients with previously untreatable cancers.

Specifications

Catalog Number	KC-3985
Cell Line Name	AGS-KRAS-G12D-Y96D-KI Cell Line
Clone Number	2A6
Host Cell Line	AGS
Description	Stable AGS clone expressing endogenous KRAS gene bearing Y96D mutations, No.2A6
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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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
In Vivo Validation	Yes

Cell Line Generation

AGS-KRAS-G12D-Y96D-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of AGS-KRAS-G12D-Y96D-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of AGS-KRAS-G12D-Y96D-KI cell line stable clone using RT-PCR sequencing.

Figure 3. Characterization of dose-response curves for KRAS inhibitors on AGS and AGS-KRAS-G12D-Y96D-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 1:3-1:6 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

Prior, I. A., Lewis, P. D., & Mattos, C. (2012). A comprehensive survey of ras mutations in cancer.Cancer Research, 72(10), 2457-2467. https://doi.org/10.1158/0008-5472.CAN-11-2612
Jänne, P. A., & Shaw, A. T. (2019). Targeting KRAS-mutant cancers with KRAS(G12C) inhibitors.New England Journal of Medicine, 381(14), 1301-1303. https://doi.org/10.1056/NEJMp1909644