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Development of a cellular biosensor system for genotoxicity detection based on Trp53 promoter
- Source :
- Human & Experimental Toxicology. 35:1102-1107
- Publication Year :
- 2016
- Publisher :
- SAGE Publications, 2016.
-
Abstract
- Objective: To develop a mouse cell biosensor system for the high-throughput genotoxicity detection of chemicals, such as environmental pollutants. Method: We developed a novel reporter vector pGL4-GFP, wherein the firefly luciferase reporter gene in the pGL4.82 vector was replaced by the green fluorescent protein (GFP) gene from the pAcGFP1-N1 vector. To construct the reporter pGL4-p53-GFP (p53 promoter linked to GFP), a fragment containing the p53 gene promoter was generated by amplifying a region from −481 to +180 of mouse genomic DNA isolated from mouse tail tissue. We developed a mouse cell biosensor system for the high-throughput genotoxicity detection of new drugs by stably integrating the reporter plasmid of pGL4-p53-GFP into the mouse embryonic fibroblast cells. Various genotoxic agents were used to treat this biosensor system. The resulting fluorescence was directly observed under a fluorescence microscope, and the GFP protein level was measured through Western blot analysis. Result: The biosensor system was treated with genotoxic agents, such as doxorubicin, cyclophosphamide, and benzo(a)pyrene. The GFP protein expression was significantly increased in cells exposed to genotoxic agents but negatively responded to the non-genotoxic agent dimethyl sulfoxide, thereby proving the specificity and sensitivity of the biosensor system. Conclusion: This novel in vitro biosensor system can be especially useful in genotoxicity detection.
- Subjects :
- 0301 basic medicine
Health, Toxicology and Mutagenesis
Blotting, Western
Genetic Vectors
Green Fluorescent Proteins
Biosensing Techniques
Biology
Toxicology
medicine.disease_cause
Cell Line
Green fluorescent protein
Mice
03 medical and health sciences
0302 clinical medicine
Plasmid
Genes, Reporter
Fluorescence microscope
medicine
Animals
Luciferase
Promoter Regions, Genetic
Reporter gene
Mutagenicity Tests
Promoter
General Medicine
Fibroblasts
Molecular biology
High-Throughput Screening Assays
030104 developmental biology
030220 oncology & carcinogenesis
Tumor Suppressor Protein p53
Biosensor
Genotoxicity
Mutagens
Plasmids
Subjects
Details
- ISSN :
- 14770903 and 09603271
- Volume :
- 35
- Database :
- OpenAIRE
- Journal :
- Human & Experimental Toxicology
- Accession number :
- edsair.doi.dedup.....02001a8315396b51a2be925d39a0eca0