GreenScreen HC is an accurate and fast in vitro human cell-based genotoxicity assay - . The TK6 host cells are p53 competent and familiar to most genetic toxicology laboratories. A patented GFP reporter system exploits the proper regulation of the GADD45a gene.
The assay delivers both high specificity and high sensitivity and detects all common mechanistic classes of genotoxin. The 96-well microplate format takes about 20 minutes to prepare and delivers results after 48 hour incubation. A single microplate is used for the simultaneous testing of 4 compounds over 9 serial dilutions. The protocol is also readily automated using standard laboratory equipment.
Biology:
GADD45a mediates the adaptive response to genotoxic stress. The patented GFP fluorescence reporter includes complex regulatory elements. The assay gives positive results for direct acting mutagens, clastogens, as well as aneugens, and topoisomerase and polymerase inhibitors. Importantly the assay gives correct negative results for non-carcinogens, many of which give misleading positive results in other in vitro tests.
Assay Protocol:
Nine, 2-fold dilutions of each compound together with positive controls are set out in the microplate and growing cells are added to each well. After incubation and measurement in a microplate reader, simple software gives automated decisions and a clear graphical output.
Validation:
A unique combination of both high specificity and high sensitivity genotoxicity assessment in a human cell line has been demonstrated.
Click here to view a number of published papers about GreenScreen HC.
Applications:
There is a compelling economic case for the use of GreenScreen HC at all stages of drug discovery from compound profiling to early lead optimisation screening, candidate selection and troubleshooting.
GADD45a:
The GADD45a-GFP GreenScreen HC genotoxicity assay monitors genotoxin-induced transcription of the GADD45a gene. GADD45a, originally identified and named by the Fornace laboratory has been implicated in the response to genome damage by genetic, biochemical, and genomic approaches. Mice lacking the gene are more prone to tumors induced by ionizing radiation and genotoxins; their lymphobasts and fibroblasts have defective nucleotide excision repair; their fibroblasts show centrosome amplification and unequal segregation of chromosomes due to multiple spindle poles, and the induction of aneuploidy. The GADD45a protein modifies DNA accessibility in damaged chromatin and associates with nuclear factors associated with cell cycle regulation. In micro-array studies, the gene is one of those most robustly induced by genotoxins. GADD45a was the first gene to be identified as a target of p53, and has key roles in cell cycle regulation and DNA repair as well as apoptosis. These studies all implicate GADD45a as a clear component of the pathways that contributes to the maintenance of genomic stability following genotoxic stress, and this is reflected in its induction by mutagens, clastogens and aneugens.
Click here to download a flyer on the GreenScreen HC assay....