KC-5102

293T-EGFR-MET-KO Cell Line

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Background of 293T-EGFR-MET-KO Cell Line

The hepatocyte growth factor receptor (MET) is a potential therapeutic target in a number of cancers, including NSCLC. In NSCLC, MET pathway activation is thought to occur through a diverse set of mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. The MET proto-oncogene was first identified in osteosarcoma cells exposed to carcinogens. Although expressed in many normal cells, MET is overexpressed in many human cancers. MET is involved in the initiation and development of various human cancers and mediates proliferation, migration and invasion. Accordingly, MET has been successfully used as a biomarker for diagnosis and prognosis, survival, post-operative recurrence, risk assessment and pathologic grading, as well as a therapeutic target. In addition, recent work indicates that inhibition of MET expression and function has potential clinical benefit.

Specifications

Catalog Number	KC-5102
Cell Line Name	293T-EGFR-MET-KO Cell Line
Clone Number	1A4
Host Cell Line	293T-EGFR-KO-1C2
Description	Stable 293T-EGFR-KO clone with human MET gene knockout, No.1A4
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	Epithelial
Subculture	Split saturated culture 1:5-1:8 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

293T-EGFR-MET-KO-1A4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-EGFR-MET-KO-1A4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-EGFR-MET-KO-1A4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-EGFR-MET-KO-1A4 cell line stable clone using FACS.

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 1:5-1:8 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

Drilon A, Cappuzzo F, Ou SI, Camidge DR. Targeting MET in Lung Cancer: Will Expectations Finally Be MET? J Thorac Oncol. 2017 Jan;12(1):15-26. doi: 10.1016/j.jtho.2016.10.014. Epub 2016 Oct 26. PMID: 27794501; PMCID: PMC5603268.
Yang X, Liao HY, Zhang HH. Roles of MET in human cancer. Clin Chim Acta. 2022 Jan 15;525:69-83. doi: 10.1016/j.cca.2021.12.017. Epub 2021 Dec 21. PMID: 34951962.