BA/F3_EGFR L858R-T790M激酶Kinase穩(wěn)定表達(dá)細(xì)胞株-BioVector NTCC保藏中心
I. Introduction
Cell Line Name：

Ba/F3_EGFR-L858R/T790M

Gene Synonyms:

Epidermal Growth Factor Receptor, ERBB1, EGFR

Host Cell:

Ba/F3

Stability: 16 passages
Application:

Anti-proliferation assay and PD assay

Freeze Medium:

90% FBS+10% DMSO

Complete Culture Medium:

RPMI-1640+10% FBS+1 ug/ml puromycin

Mycoplasma Status:

Negative


II.Background
EGFR is widely recognized for its importance in cancer. Amplification and mutations have been shown to be driving events in many cancer types. Its role in non-small cell lung cancer, glioblastoma and basal-like breast cancers has spurred many research and drug development efforts. Tyrosine kinase inhibitors have shown efficacy in EGFR amplfied tumors, most notably gefitinib and erlotinib. Mutations in EGFR have been shown to confer resistance to these drugs, particularly the variant T790M, which has been functionally characterized as a resistance marker for both of these drugs. The later generation TKI's have seen some success in treating these resistant cases, and targeted sequencing of the EGFR locus has become a common practice in treatment of non-small cell lung cancer. Overproduction of ligands is another possible mechanism of activation of EGFR. ERBB ligands include EGF, TGF-a, AREG, EPG, BTC, HB-EGF, EPR and NRG1-4 (for detailed information please refer to the respective ligand section). In ligand-activated cancers, Cetuximab appears to be more effective than tyrosine-kinase inhibitors.


III. Representative Data
1. WB of EGFR-L858R/T790M expression


Figure 1. WB of EGFR Expression
Lane 1: Negative control
Lane 2: EGFR-WT
Lane 3: EGFR-L858R/T790M
Lane 4: EGFR-L858R/T790M/C797S

2. Sanger sequencing


Figure 2. Sanger Sequencing of EGFR-L858R/T790M

3. Anti-proliferation assay



Figure 3. Anti-proliferation assay of three reference compounds on the Ba/F3_EGFR-L858R/T790M Stable Cell Line


IV. Thawing

Thawing: Protocol
1. Remove the vial from liquid nitrogen tank and thaw cells quickly in a 37°C water-bath.
2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol and transfer the cells to a 15 ml centrifuge tube containing 9 ml of complete growth medium.
3. Pellet cells by centrifugation at 200 x g force for 5 min, and discard the medium.
4. Resuspend the cells in complete growth medium.
5. Add 10 ml of the cell suspension in a 10 cm dish.
6. Add promycin to a concentration of 1 μg/ml the following day.
Kinase細(xì)胞株



現(xiàn)代新藥研發(fā)的關(guān)鍵首先是尋找，確定和制備藥物作用靶點(diǎn)。在500多個(gè)已發(fā)現(xiàn)的藥物靶點(diǎn)里中，GPCR，Ion Channel，Kinase使用的最為廣泛。




激酶(kinase)是一類從高能供體分子（如ATP）轉(zhuǎn)移磷酸基團(tuán)到特定靶分子（底物）的酶；這一過(guò)程謂之磷酸化。許多腫瘤的發(fā)生是由某些與生長(zhǎng)相關(guān)的“激酶”發(fā)生突變導(dǎo)致異?；罨鸬?，因而針對(duì)這些突變激酶的抑制劑能夠有效抑制這些激酶的活性，從而達(dá)到抑制癌細(xì)胞增長(zhǎng)的目的。
BioVector NTCC質(zhì)粒載體菌種細(xì)胞蛋白抗體基因保藏中心

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