Protocol

Genetic Toxicology

Volume 817 of the series Methods in Molecular Biology pp 231-250

Date:

The GADD45a-GFP GreenScreen HC Assay

  • Richard M. WalmsleyAffiliated withGentronix Ltd Email author 
  • , Matthew TateAffiliated withGentronix Ltd., University of Manchester

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Abstract

Mutagens, clastogens, and aneugens cause increased expression of the human GADD45a gene. This has been exploited in the GreenScreen HC genotoxicity assay in which the gene’s expression is linked to the expression of green fluorescent protein (GFP). The host for the reporter construct is the human lymphoblastoid cell line TK6. It was chosen for its growth as a cell suspension, which allows simple pipette transfers, and for its wild-type p53 competent status. P53 is required for proper GADD45a expression, and more generally for genome stability. TK6 is a karyotypically stable cell line.

The GreenScreen assays were designed to facilitate screening, and this is reflected in its microplate format and low compound requirement. Protocols are available for testing with and without S9 as a source of exogenous metabolic activation. Data is collected either spectrophotometrically or by flow cytometry, and a simple spreadsheet converts raw data into dose–response curves, and provides a statistically significant positive or negative result. Extensive validation has demonstrated that in contrast to other in vitro mammalian genotoxicity assays, the GADD45a assays have both high sensitivity and specificity – they very rarely produce misleading positive results.

Key words

GADD45a GADD45a-GFP GreenScreen HC Screening Genotoxicity