KC-5710

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

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

c-Met, also named as hepatocyte growth factor receptor (HGFR), is a single pass tyrosine kinase receptor, which is mainly expressed on the cells of epithelial origin, and play essential role in embryonic development, organogenesis and wound healing. HGF and its splicing isoform are the only know ligands of cMet. Abnormal activation of cMet due to overexpression of Met or its ligands, fusion mutation as well as exon14 skipping have been implicated in oncogenesis. 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-5710
Cell Line Name	293T-EGFR-cMET-KO-mouse-cMET Cell Line
Clone Number	2#
Host Cell Line	293T-EGFR-cMET-KO
Description	Stable 293T EGFR cMET KO cell line expressing exogenous mouse cMET genes
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 + 1ug/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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 cMET KO mouse cMET cell line was generated using a lentiviral vector expressing the mouse cMET sequence.

Characterization

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

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

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1ug/mL Puromycin)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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Saffroy, R. et al. MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung. Oncotarget 8, 42428ÿ42437 (2017).
2. Peschard, P. & Park, M. From Tpr-Met to Met, tumorigenesis and tubes. Oncogene 26, 1276ÿ1285 (2007).