Title:
A CELL BASED SYSTEM FOR RAPID DETECTION AND QUANTIFICATION OF GENOTOXICITY
Document Type and Number:
WIPO Patent Application WO/2011/105968
Kind Code:
A3
Abstract:
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.
Inventors:
FILIPIČ METKA (SI)
LAH TURNŠEK TAMARA (SI)
HRELJAC IRENA (SI)
ČEMAŽAR MAJA (SI)
SERŠA GREGOR (SI)
ŽAGER VALERIJA (SI)
KAMENŠEK URŠKA (SI)
LAH TURNŠEK TAMARA (SI)
HRELJAC IRENA (SI)
ČEMAŽAR MAJA (SI)
SERŠA GREGOR (SI)
ŽAGER VALERIJA (SI)
KAMENŠEK URŠKA (SI)
Application Number:
PCT/SI2011/000010
Publication Date:
July 02, 2015
Filing Date:
February 22, 2011
Export Citation:
Assignee:
NACIONALNI INŠTITUT ZA BIOLOG (SI)
FILIPIČ METKA (SI)
LAH TURNŠEK TAMARA (SI)
HRELJAC IRENA (SI)
ČEMAŽAR MAJA (SI)
SERŠA GREGOR (SI)
ŽAGER VALERIJA (SI)
KAMENŠEK URŠKA (SI)
FILIPIČ METKA (SI)
LAH TURNŠEK TAMARA (SI)
HRELJAC IRENA (SI)
ČEMAŽAR MAJA (SI)
SERŠA GREGOR (SI)
ŽAGER VALERIJA (SI)
KAMENŠEK URŠKA (SI)
International Classes:
G01N33/50
Domestic Patent References:
| WO2004078985A1 | 2004-09-16 |
Other References:
CEMAZAR M ET AL: "Construction of EGFP Expressing HepG2 Cell Line Using Electroporation", 11TH INTERNATIONAL CONGRESS OF THE IUPESM. PROCEEDINGS WORLD CONGRESS ON MEDICAL PHYSICS AND BIOMEDICAL ENGINEERING. SPECIAL TOPICS AND WORKSHOPS. (IFMBE 2009) SPRINGER-VERLAG BERLIN, GERMANY, 2009, pages 128 - 131, XP009148752, ISBN: 978-3-642-03894-5
ZAGER V ET AL: "Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression", RADIOLOGY AND ONCOLOGY 20100301 SLOVENIAN MEDICAL SOCIETY / CROATIAN MEDICAL ASSOCIATION SVN LNKD- DOI:10.2478/V10019-010-0010-3, vol. 44, no. 1, 1 March 2010 (2010-03-01), pages 42 - 51, XP002638472, ISSN: 1318-2099
ZAGER V ET AL: "Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression", RADIOLOGY AND ONCOLOGY 20100301 SLOVENIAN MEDICAL SOCIETY / CROATIAN MEDICAL ASSOCIATION SVN LNKD- DOI:10.2478/V10019-010-0010-3, vol. 44, no. 1, 1 March 2010 (2010-03-01), pages 42 - 51, XP002638472, ISSN: 1318-2099
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