Green fluorescent protein (GFP) as a direct biosensor for mutation detection: Elimination of false-negative errors in target gene expression

Abstract

A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(±)-7 p,8 a-dihydroxy-9 a,10 a-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10–500 μM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0–8.59 μM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE–AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.