KC-5146

293T-NFAT-Luc2-cMET-KO-EGFR Cell Line

57081

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Background of 293T-NFAT-Luc2-cMET-KO-EGFR Cell Line

EGFR (Epidermal Growth Factor Receptor) is a Protein Coding gene. Diseases associated with EGFR include Neonatal Nephrocutaneous Inflammatory Syndrome and Lung Cancer. Among its related pathways are Apoptotic Pathways in Synovial Fibroblasts and Signaling by EGFR in Cancer.MET (MET Proto-Oncogene, Receptor Tyrosine Kinase) is a Protein Coding gene. Diseases associated with MET include Renal Cell Carcinoma, Papillary, 1 and Arthrogryposis, Distal, Type 11. Among its related pathways are Apoptotic Pathways in Synovial Fibroblasts and GPCR Pathway. Gene Ontology (GO) annotations related to this gene include transferase activity, transferring phosphorus-containing groups and protein tyrosine kinase activity.

Specifications

Catalog Number	KC-5146
Cell Line Name	293T-NFAT-Luc2-cMET-KO-EGFR Cell Line
Clone Number	1B1
Host Cell Line	293T-cMET-KO-EGFR
Description	Stable 293T-cMET-KO-EGFR cell line expressing exogenous luciferase under the control of NFAT signaling pathway.
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 + 150μg/mL Hygromycin B + 1μg/mL Puromycin
Selection Marker	Hygromycin B, Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1 : 10 every 3 days; seed out at about 1 × 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-NFAT-Luc2-cMET-KO-EGFR Cell Line was generated using a lentiviral vector expressing the NFAT and luciferase sequence.

Characterization

Figure1: Characterization of EGFR overexpression and cMET Knock out in 293T cell line stable clones using FACS.

Figure2: Characterization of cMET Knock out in 293T cell line stable clones using RT-PCR sequencing.

Figure3: Characterization of EGFR overexpression in 293T cell line stable clones using RT-PCR sequencing.

Figure4: 293T-NFAT-Luc2-cMET-KO-EGFR cells were seeded into 96-well plates, treated with EGF for 16 hours, and then read out using Bright-Glo Detection System.

Figure5: 293T-NFAT-Luc2-cMET-KO-EGFR cells were seeded into 96-well plates, treated with Cetuxima(Cat#KB-1106, Kyinno) for 1 hours, and then treated with EGF in the concentration of 100ng/mL for 16 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin B and 1μg/mL Puromycin.) 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 : 10 every 3 days; seed out at about 1 × 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

1. Campbell P, Morton PE, Takeichi T, Salam A, Roberts N, Proudfoot LE, Mellerio JE, Aminu K, Wellington C, Patil SN, Akiyama M, Liu L, McMillan JR, Aristodemou S, Ishida-Yamamoto A, Abdul-Wahab A, Petrof G, Fong K, Harnchoowong S, Stone KL, Harper JI, Irwin McLean WH, Simpson MA, Parsons M, McGrath JA. Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR. J Invest Dermatol. 2014 Oct;134(10):2570-2578. doi: 10.1038/jid.2014.164. Epub 2014 Apr 1. PMID: 24691054; PMCID: PMC4090136.
2. Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 2005 Feb 24;352(8):786-92. doi: 10.1056/NEJMoa044238. PMID: 15728811.
3. Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, Scherer SW, Zhuang Z, Lubensky I, Dean M, Allikmets R, Chidambaram A, Bergerheim UR, Feltis JT, Casadevall C, Zamarron A, Bernues M, Richard S, Lips CJ, Walther MM, Tsui LC, Geil L, Orcutt ML, Stackhouse T, Lipan J, Slife L, Brauch H, Decker J, Niehans G, Hughson MD, Moch H, Storkel S, Lerman MI, Linehan WM, Zbar B. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet. 1997 May;16(1):68-73. doi: 10.1038/ng0597-68. PMID: 9140397.
4. Zhou H, Lian C, Wang T, Yang X, Xu C, Su D, Zheng S, Huang X, Liao Z, Zhou T, Qiu X, Chen Y, Gao B, Li Y, Wang X, You G, Fu Q, Gurnett C, Huang D, Su P. MET mutation causes muscular dysplasia and arthrogryposis. EMBO Mol Med. 2019 Mar;11(3):e9709. doi: 10.15252/emmm.201809709. PMID: 30777867; PMCID: PMC6404111.