Biochemical and Biophysical Research Communications
Chemical stress sensitive luminescent human cells: Molecular biology approach using inducible Drosophila melanogaster hsp22 promoter
Section snippets
Materials and methods
Chemicals. Heavy metals and chemicals tested (CdCl2, Cd(NO3)2, NaAsO2, alachlore, fentine acetate, thiram and maneb) were obtained from Sigma–Aldrich (France).
Construction of reporter vectors. A set of reporter vectors, derived from the pGL3-Enhancer vector (Promega, Madison, USA), Fig. 1A, containing the Photinus pyralis firefly luciferase (luc) reporter gene, was constructed. In a first step, a 620 bp HindIII fragment containing Drosophila melanogaster hsp22 promoter was excised from the
Heat shock and chemical induction of the hsp22 Drosophila melanogaster promoter stress induction in HeLa cells
The efficiency of induction was measured as the ratio of signal-to-noise calculated asThe induction of hsp22 full-length promoter by heat shock was used to determine the optimal experimental conditions to study light emission in our cell system. For that purpose, HeLa cells transiently transfected with the expression vector, pGL3-pd22-−400-luc, carrying the luc gene under the control of the full-length Drosophila melanogaster
Conclusions
The aim of this study was to develop a simple whole-cell bioassay for pollution detection. For that purpose, bioluminescent eukaryotic cells were genetically modified using a mammalian vector carrying the firefly luciferase gene (luc) reporter gene, under the control of the Drosophila melanogaster hsp22 stress inducible promoter. Such a promoter responds to cellular stress usually induced by heat shock, but also by chemical agents like heavy metals, genotoxic agents, and endocrine disruptors.
Acknowledgments
Part of this work was supported by the Région Rhône-Alpes (Thématiques Prioritaires), the Université Claude Bernard Lyon1 (Bonus Qualité Recherche) and the European Commission, (MENDOS project/QLK4-CT2002-02323).
References (31)
- et al.
Comparative study of analytical methods involving gas chromatography-mass spectrometry after derivatization and gas chromatography-tandem mass spectrometry for the determination of selected endocrine disrupting compounds in waste waters
J. Chromatogr. A.
(2004) - et al.
Toxicity and genotoxicity of industrial soils polluted by polycyclic aromatic hydrocarbons (PAHs)
Org. Geochem.
(1999) - et al.
Issues underlying use of biosensors to measure metal bioavailability
Ecotoxicol. Environ. Saf.
(2003) - et al.
Activation of retinoic acid receptor-dependant transcription by organochlorine pesticides
Toxicol. Appl. Pharmacol.
(2005) - et al.
Immobilized cells used for detection and analysis
Curr. Opin. Biotechnol.
(1994) - et al.
A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection
Biosens. Bioelectron.
(2005) - et al.
Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation
Mol. Cell. Endocrinol.
(2001) - et al.
A two-stage minibioreactor system for continuous toxicity monitoring
Biosens. Bioelectron.
(1999) - et al.
Versatile biosensor vectors for detection and quantification of mercury
FEMS Microbiol. Lett.
(2000) - et al.
Aging-specific expression of Drosophila hsp22
Dev. Biol.
(1999)
Differential expression of heat shock protein in Drosophila embryonic cells following metal ion exposure
Exp. Cell. Res.
Nucleotide sequence analysis of the Drosophila melanogaster small heat shock gene cluster at locus 67B
J. Mol. Biol.
Ecdysterone selectively stimulates the expression of a 23,000 dalton heat-shock protein β-galactosidase hybrid gene in cultured Drosophila cells
Dev. Biol.
The kinetics of HIV-1 long terminal repeat transcriptional activation resemble those of hsp70 promoter in heat-shock treated HeLa cells
FEBS Lett.
Cited by (11)
Recent progress on cell-based biosensors for analysis of food safety and quality control
2019, Biosensors and BioelectronicsCitation Excerpt :The biosensors used to detect heavy metals in food are summarized in Table 2. A cell bioassay has been developed for total toxicity testing of liquid samples (Mandon et al., 2005a, 2005b). The assay is based on the induction of bioluminescent activity in genetically manipulated mammalian cells.
Development of cadmium specific recombinant biosensor and its application in milk samples
2017, Sensors and Actuators, B: ChemicalCitation Excerpt :For that purpose, a DNA sensing element that responds to chemical stress agents (heavy metals, genotoxic agents, and endocrine-disrupting chemicals) was transfected in HeLa cells. Such element was designed to direct the expression of a reporting gene (firefly luciferase) through the activation of Drosophila melanogaster hsp22 promoter [4]. Although, there is many other stress causing reasons such as genotoxicity, endocrine disruption etc. to induce the luc gene expression, therefore this approach is not suitable in the term of specific detection of cadmium metal ion.
Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production
2013, Biosensors and BioelectronicsCitation Excerpt :The signal intensity per spot was calculated as the median intensity for all pixels included in a circular feature defining the spot and corrected using a local background evaluation. Fluorescent HeLa cell lines (EGFP-HeLa) were obtained by stable transfection of the pEGFP-C1 vector (Clontech, Heidelberg, Germany) using lipofectamine reagent from life/technologies/Invitrogen (Cergy Pontoise, France) and following a previously published protocol (Mandon et al., 2005). This EGFP constitutive expression vector contains the neomycin phosphotransferase gene for selection of stably transfected clones.
Portable and handheld cell-based biosensors
2017, Portable Biosensors and Point-of-Care SystemsMaterial surface engineering for multiplex cell culture in microwell
2014, Journal of Materials Science