U2OS lmmil 1-TUBA1B - ACTB,

Application

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This product is a human U2OS cell line in which three genomic loci - LMNB1, TUBA1B, and ACTB - have been endogenously tagged with fluorescent protein genes for Blue Fluorescent Protein (BFP), Green Fluorescent Protein (GFP), and Red Fluorescent Protein (RFP), respectively, using CompoZr^® zinc finger nuclease (ZFN) technology. Integration resulted in endogenous expression of: 1) the fluorescent fusion protein, BFP-laminB1, easily detected in the nuclear envelope; 2) the fluorescent fusion protein, GFP-α-tubulin, that polymerizes to form characteristic patterns of microtubules; 3) the fluorescent fusion protein, RFP-β-actin, that polymerizes to form characteristic patterns of actin fibers. Imaging of the cell line shows fluorescent labeling of the three proteins having normal cellular distribution patterns which makes the cell line ideal for high content screening to identify compounds that modulate cellular activity. This stable cell line was expanded from a single cell clone. The LMNB1, TUBA1B, and ACTB gene regulation and corresponding protein function are preserved in contrast to cell lines exhibiting overexpression from an exogenous promoter.

Biochem/physiol Actions

Nuclear lamin B1, encoded by the 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. Thus, LMNB1 can be used as a potential biomarker for HCC.Tubulin α 1b (TUBA1B) is a microtubule protein, which plays a vital role in dynamic process of polymerization and depolymerization during cell replication and division. TUBA1B is overexpressed in hepatocellular carcinoma (HCC) tumor tissues and proliferating HCC cells. This upregulated expression of the gene leads to poor prognosis and chemotherapy resistance in HCC.Actin β (ACTB) is an abundant cytoskeletal housekeeping protein. The protein is expressed in the nucleus and controls gene expression, cell division and proliferation. ACTB is generally used as a reference gene in measuring expression levels in tumors.

Features and Benefits

Zinc Finger Nuclease (ZFN)-mediated targeted integration of three different fluorescent tags: LMNB1 is BFP-tagged on chromosome 5q23.2-q31.1, TUBA1B is GFP-tagged on chromosome 12q13.12 and ACTB is RFP-tagged on chromosome 7p22.1 to create a cell line exhibiting stable expression of all three transgenes.The U2OS cells are adherent, with a doubling time of approx. 29 hours.

General description

U2OS LMNB1-TUBA1B-ACTB are bone osteosarcoma cells from a human 15 year old caucasion female having three distinct ZFN modifications creating a BFP-tagged LMNB1 transgene, a GFP-tagged TUBA1B transgene and a RFP-tagged ACTB transgene expressed from their endogenous gene locus.This cell line was derived from ATCC Catalog No. HTB-96.

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 McCoy′s 5A Medium Modified, Cat. No. M9309. 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%.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.