KC-5785

Ba/F3-KRAS-G12D-A59G Cell Line

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Background of Ba/F3-KRAS-G12D-A59G 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. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-5785
Cell Line Name	Ba/F3-KRAS-G12D-A59G Cell Line
Clone Number	11#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous KRAS gene bearing G12D-A59G 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	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	NA
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 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 20 hours
Mycoplasma Status	Negative

Cell Line Generation

Ba/F3-KRAS-G12D-A59G Cell Line was generated using retrovirus vector expressing KRAS-G12D-A59G sequence.

Characterization

Figure 1: Characterization of KRAS mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Ba/F3 cells expressing KRAS mutant were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS)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 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI-1640 + 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.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