A549 Cells GFP-EGFR,

Application

A549 Cells GFP-EGFR is an A549 cell line in which the genomic EGFR gene has been endogenously tagged with a Green Fluorescent Protein gene (GFP) using CompoZr^® Zinc Finger Nuclease technology. Integration resulted in endogenous expression of the fusion protein in which GFP is attached to the C-terminus of EGFR. Fluorescence imaging shows characteristic EGFR membrane expression. Upon the addition of EGF, the cell line shows redistribution of EGFR from the cell membrane to endosomes, making it useful for high content screening of compounds. For example, the redistribution can be abolished by a selective inhibitor of EGFR, Tyrphostin AG 1478. This stable cell line was expanded from a single clone. The target’;s gene regulation and corresponding protein function are preserved in contrast to cell lines with overexpression via an exogenous promoter.

Features and Benefits

Zinc Finger Nuclease (ZFN)-mediated targeted integration of a fluorescent GFP tag into the last coding exon of the EGFR gene on chromosome 7p11.2 to create a cell line exhibiting stable expression of the transgene tagged with GFP on the C-terminus of the protein.These A549 cells are adherent with a doubling time of approx. 22 hours.

General description

Glucocorticoid is necessary for A549 cell induction to respond to OSM (oncostatin M), interleukin (IL-6) and fibroblast conditioned medium. The EGFR gene is mapped to human chromosome 7p11.2 and encodes a 170kDa receptor protein belonging to the HER family of receptor tyrosine kinases. Egfr is localized to epithelial cell surface.

A549 GFP-EGFR are lung carcinoma, epithelial cells from a human 58 year old male caucasion having a ZFN modification creating an EGFR-GFP transgene expressed from the endogenous EGFR gene locus.This cell line was derived from ATCC catalog No. CCL-185.

Legal Information

CompoZr is a registered trademark of Sigma-Aldrich Co. LLC

Preparation Note

Media Renewal changes two to three times per week.Rapidly thaw vial by gentle agitation in 37°C water bath (~2 minutes), keeping vial cap out of the water. Decontaminate with 70% ethanol, add 9 mL culture media and centrifuge 125 x g (5-7 minutes). Resuspend in complete culture media and incubate at 37°C in a 5% CO2 atmosphere.Subculture Ratio: approx. 1:3-1:6The base medium for this cell line is RPMI-1640, Cat. No. R0883. To make the complete growth medium, add the following components to the base medium: fetal bovine serum, Cat. No. F2442, to a final concentration (v/v) of 10% and L-glutamine, Cat. No. G7513, at a final concentration of 2 mM.Cell freezing medium-DMSO 1X, Cat. No. C6164.

Quality

Tested for Mycoplasma, sterility, post-freeze viability, short terminal repeat (STR) analysis for cell line identification, cytochrome oxidase I (COI) analysis for cell line species confirmation.