KC-5580

293T-EGFR-cMET-KO-mouse-EGFR Cell Line

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Background of 293T-EGFR-cMET-KO-mouse-EGFR Cell Line

EGFR (Epidermal Growth Factor Receptor) and cMET (Mesenchymal-epithelial transition factor) are receptor tyrosine kinases that drive cell proliferation, survival, and metastasis. EGFR is also known as ERBB1 or HER1, while cMET is also known as HGFR. Co-activation of EGFR and cMET is frequently observed in various cancers, including non-small cell lung cancer, where cMET amplification mediates resistance to EGFR-TKIs. Knockout of both receptors in 293T cells eliminates endogenous signaling, allowing functional study of exogenous mouse EGFR without cross-talk. Mouse EGFR shares high homology with human EGFR, making this cell line suitable for evaluating species‑specific EGFR inhibitors and studying resistance mechanisms.

Specifications

Catalog Number	KC-5580
Cell Line Name	293T-EGFR-cMET-KO-mouse-EGFR Cell Line
NCBI/UniProt Accession Number	NM_207655.2
Clone Number	3#
Host Cell Line	293T-EGFR-cMET-KO
Description	Stable 293T cell line with knockout of endogenous EGFR and cMET, and expressing exogenous mouse EGFR.
Quantity	One vial 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

The 293T-EGFR-cMET-KO-mouse-EGFR cell line was generated using CRISPR/Cas9 to knock out EGFR and cMET, followed by lentiviral transduction of mouse EGFR sequence.

Characterization

Figure 1: Characterization of mouse EGFR overexpression in the 293T-EGFR-cMET-KO-mouse-EGFR stable clone using FACS.

Figure 2: Characterization of mouse EGFR overexpression in the 293T-EGFR-cMET-KO-mouse-EGFR stable clone using PCR sequence.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Passiglia, Francesco et al. “The role of cMet in non-small cell lung cancer resistant to EGFR-inhibitors: did we really find the target?.” Current drug targets vol. 15,14 (2014): 1284-92. doi:10.2174/138945011514141216092739
2. Seo, Donghyun, and Jun Hyeok Lim. “Targeted Therapies for EGFR Exon 20 Insertion Mutation in Non-Small-Cell Lung Cancer.” International journal of molecular sciences vol. 25,11 5917. 29 May. 2024, doi:10.3390/ijms25115917
3. Sabbah, Dima A et al. “Review on Epidermal Growth Factor Receptor (EGFR) Structure, Signaling Pathways, Interactions, and Recent Updates of EGFR Inhibitors.” Current topics in medicinal chemistry vol. 20,10 (2020): 815-834. doi:10.2174/1568026620666200303123102
4. Machiraju, Devayani, and Jessica C Hassel. “Targeting the cMET pathway to enhance immunotherapeutic approaches for mUM patients.” Frontiers in oncology vol. 12 1068029. 24 Jan. 2023, doi:10.3389/fonc.2022.1068029

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.