SKOV3 Cells GFP-HER2 RFP-EGFR

NA.81

SKOV3 GFP-HER2 RFP-EGFR are ovarian adenocarcinoma cells from a human 64 year old caucasion female having two ZFN modifications - a HER2-GFP transgene and a EGFR-RFP transgene both expressed from their corresponding endogenous gene locus.

This cell line was derived from ATCC Catalog No. HTB-77.

This product is a human SKOV3 cell line in which the genomic HER2 gene has been endogenously tagged with a Green Fluorescent Protein (GFP) gene and the genomic EGFR gene with a Red Fluorescent Protein (RFP) gene using CompoZr^® Zinc Finger Nuclease technology. Integration resulted in endogenous expression of a fusion protein in which GFP is attached to the C-terminus of HER2 and of a fusion protein in which RFP is attached to the C-terminus of EGFR. Flourescence imaging shows chacteristic HER2 and 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.

Zinc Finger Nuclease (ZFN)-mediated targeted integrations of a fluorescent GFP tag into the last coding exon of the HER2 gene on chromosome 17q21.1 and an RFP tag into the last coding exon of the EGFR gene on chromosome 7p11.2 to create a cell line exhibiting stable expression of both transgenes - each tagged with the corresponding fluorescent reporter on the C-terminus of the protein.

These SKOV3 cells are adherent with a doubling time of approx. 48 hours.

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.

Media Renewal changes two to three times per week.

Rapidly thaw vial by gentle agitation in 37℃ 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℃ in a 5% CO2 atmosphere.

Subculture Ratio: approx. 1:3-1:6

The base medium for this cell line is McCoy′s 5A Medium, Cat. No. M8403. 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 1.5 mM.

Cell freezing medium-DMSO 1X, Cat. No. C6164.

CompoZr is a registered trademark of sigma-aldrich Co. LLC

human female ovary (Source Disease: Ovarian adenocarcinoma)

200

131550
164870

−196℃

human ... EGFR(1956), ERBB1(1956), ERBB2(2064), HER2(2064)