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In vitro reporter gene assays

Dr. Ludek Blaha

Masaryk University, RECETOX, Brno


Within the project, a set of specific in vitro reporter gene assays will be used. The assays have been developed to screen for potential toxicity of chemicals, their mixtures and contaminated samples, based on the specific biochemical mechanisms (e.g. PCBs with dioxin-like effects, endocrine disrupters or mutagens/genotoxins). The tests inform about the overall toxic potential of the studied samples. The bioassays are based on genetically modified cell lines, which have been stably transfected with specific reporter genes (e.g. luciferase from the firefly). Reporter genes are induced and translated in the presence of specifically acting compounds (e.g. estrogens, androgens etc.), and the enzymatic activity of the reporter protein is easily determined (e.g. measuring bioluminescence). The battery of bioassays then provide a complex overview of the overall toxicity of the contaminated sample (e.g. surface water, effluent, sediments, soils etc.)

Following bioassays will be used within the project:

- Dioxin-like toxicity - rat hepatoma cell line H4IIE.luc, which determines dioxin-like compounds (e.g. PCBs, dioxins etc.) by measuring luciferase activity under the control of AhR (arylhydrocarbon receptor).

- Estrogenic effects - potential effects of endocrine disruptors causing feminizations - are determined with human breast carcinoma cell line MVLN, stably trasfected with luciferase reporter gene under the control of ER (estrogen receptor)

- Androgenic and anti-androgenic effects - will be determined using reporter gene assays with yeasts or breast carcinoma cell line MDAkB

- Genotoxic effects will be tested with the SOS chromotest. The assay uses bacteria Escherichia coli transfected with beta-galactosidase gene, which is induced along with the SOS repair enzymes responding to previous DNA damage

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