A549 GFP-CTNNB1 RFP-LMNB1 are lung carcinoma cells from a human 58 year old caucasian male having two distinct ZFN modifications creating two fluorescent transgenes each expressed from their endogenous gene locus.
This cell line was derived from ATCC Catalog No. CCL-185.
This product is a human A549 cell line in which the genomic CTNNB1 gene has been endogenously tagged with the 3XFLAG™ epitope linked to a Green Fluorescent Protein (GFP) gene and the LMNB1 gene has been endogenously tagged with a Red Fluorescent Protein (RFP) gene using CompoZr® zinc finger nuclease (ZFN) technology. Integration resulted in endogenous expression of the fusion proteins in which 3XFLAG-GFP is attached to the N-terminus of CTNNB1 and RFP is attached to the N-terminus of LMNB1. Fluorescence imaging shows CTNNB1 expression throughout the cell and LMNB1 expression localized to the nucleus. Upon activation with LiCl/leptomycin B or the Wnt3a ligand, the proteolysis of CTNNB1 is reduced so that translocation into the nucleus can be observed, making the cell line useful for high content screening to identify compounds that modulate this redistribution. The stable cell line was expanded from a single cell clone. CTNNB1 and LMNB1 gene regulation and corresponding protein function are preserved in contrast to cell lines exhibiting overexpression from an exogenous promoter.
β-catenin encoded by CTNNB1 gene is a multifunctional protein. It facilitates cell-cell adhesion differentiation and epithelial membrane transition (EMT). In addition, it plays a vital role in the WNT signaling pathway. Mutation in the gene leads to colorectal carcinomas. Nuclear lamin B1 encoded by LMNB1 gene is an intermediate filament protein of the nuclear envelope. The protein exhibits transcriptional co-regulatory activity and plays a key role in DNA replication, cellular aging and stress responses. Overexpression of the gene has been observed in hepatocellular carcinoma (HCC) patients. Thus, LMNB1 can be used as a potential biomarker for HCC.
Zinc Finger Nuclease (ZFN)-mediated targeted integrations of a fluorescent 3XFLAG-GFP just behind the ATG start codon of the CTNNB1 gene on chromosome 3p22.1 and a fluorescent RFP tag just behind the ATG start codon of the LMNB1 gene on chromosome 5q23.2 to create a cell line exhibiting stable expression of the two transgenes.
The A549 cells are adherent, with a doubling time of approx. 22 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°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. 1:3-1:8
The base medium for this cell line is F12 Ham Medium, Cat. No. N4888. To make the complete growth medium, add the following components to the base medium: fetal bovine serum, Cat. No. F4135, 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.
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CompoZr is a registered trademark of Sigma-Aldrich Co. LLC