A CELL BASED SYSTEM FOR RAPID DETECTION AND QUANTIFICATION OF GENOTOXICITY

摘要

The cell system for rapid detection and determination of genotoxicity is a biosensor cell system with metabolicaly competent human cells, stable transfected with reporter gene, which is operationally linked to the promoter of gene, which responds to DNA damage, and solves the problem of rapid and accurate detection and quantification of genotoxic substances and other samples. In the preferred embodiment of this invention metabolicaly competent human cells HepG2 cells stable transfected with reporter gene, preferred the gene that encodes the fluorescent protein DsRed2, and a promoter that responds to DNA damage, preferred the p21 gene promoter are used. The vector construct by which the HepG2 cells are transfected is of composed of coding sequence of nucleotides that encode for DsRed protein that is operatively linked in cis configuration to promoter region of p21 gene. The method for genotoxicity determination consists of the following steps: contact HepG2-p21-DsRed cells with tested chemical or sample, incubation at 37oC/ 5% CO2, measurement of the fluorescence of the expressed protein DsRed with a microtiter plate reader at 24 and 48 hour of exposure, determination of cells density by MTT assay, and calculation of the normalized values of induced fluorescence, which are qualitative and quantitative indicator of genotoxic activity of the tested chemical or sample.