title: Development of a cellular biosensor system for genotoxicity detection based on Trp53 promoter.
abstract: OBJECTIVE
  To develop a mouse cell biosensor system for the high-throughput genotoxicity detection of chemicals, such as environmental pollutants.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  This novel in vitro biosensor system can be especially useful in genotoxicity detection.
authors.affiliation: Laboratory of Molecular Genetics of Aging and Tumor, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China.
authors.forename: L
authors.initials: L
authors.lastname: Siqian
authors.affiliation: Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, Yunnan, China.
authors.forename: S
authors.initials: S
authors.lastname: Lei
authors.affiliation: Laboratory of Molecular Genetics of Aging and Tumor, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China yingluo@kmust.edu.cn.
authors.forename: L
authors.initials: L
authors.lastname: Ying
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057166:High-Throughput Screening Assays; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000633:toxicity*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D009153:Mutagens; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins
keywords: Genotoxcity; green fluorescence protein (GFP); mouse embryonic fibroblast cells; p53