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DNA damage sensors

IP.com Disclosure Number: IPCOM000199963D
Publication Date: 2010-Sep-22
Document File: 3 page(s) / 36K

Publishing Venue

The IP.com Prior Art Database

Abstract

A reporting system is envisaged that will enable real-time measurement of a cell's response to drugs and environmental factors. The underlying principle would be to create a stable transfection in a host mammalian cell system. The cells would constitutively express matched sets of engineered relatively short-lived fluorescent protein sensors. In the basic format, a double construct can be envisaged, comprising 1) a Red fluorescent protein (RFP), configured to produce a signal decrease over a short-term and 2) green fluorescent protein (GFP), configured to produce a signal increase over a longer term The above sensors, in addition to a nuclear Hoechst (blue) stain, would temporally measure cellular lesions, as well as transcriptional blocks and repair mechanisms, upon exposure of target cells to genotoxic challenges. The nuclear stain would allow measurement of nuclear damage by assessment of micronuclei content, for example.

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Cellular toxicity caused by drugs and environmental factors: Proposed double- and triple construct sensors enabling real-time monitoring of DNA-damage lesions and subsequent DNA repair in cells

Keywords

Cellular toxicity; toxicity sensors; genotoxic sensors

Abstract

A reporting system is envisaged that will enable real-time measurement of a cell’s response to drugs and environmental factors. The underlying principle would be to create a stable transfection in a host mammalian cell system. The cells would constitutively express matched sets of engineered relatively short-lived fluorescent protein sensors. In the basic format, a double construct can be envisaged, comprising 1) a Red fluorescent protein (RFP), configured to produce a signal decrease over a short-term and 2) green fluorescent protein (GFP), configured to produce a signal increase over a longer term

The above sensors, in addition to a nuclear Hoechst (blue) stain, would temporally measure cellular lesions, as well as transcriptional blocks and repair mechanisms, upon exposure of target cells to genotoxic challenges. The nuclear stain would allow measurement of nuclear damage by assessment of micronuclei content, for example.

Background

There is still a requirement to supplement the established Ames test (Salmonella mutagenicity) with a trustworthy test for mammalian cell-based genetic toxicity, and to generally increase the throughput for the screening of environmental chemicals and putative drugs. In an attempt to address this requirement, “Live-dead” type cellular assays have been described using cell imaging techniques coupled to image analysis software, albeit with a limited number of sensors. It would therefore be useful to create a cell-based system to allow real-time measurement of a multiple set of genotoxicity sensors within a cell, particularly early-event DNA lesions and subsequent repair processes.

Of the fluorescent proteins (FPs) used for cellular analysis, green fluorescent protein (GFP) has proved to be a useful reporter for gene toxicity studies due to its brightness, stability (t1/2 26h) and low toxicity. The long half-life of GFP however, precludes its use for study for more dynamic studies of gene expression and toxicity.

Potentially short-lived “destabilised” GFP variants have been described in the literature.

For example, Kitsera et al (1) describe construction and study of a GFP-ornithine decarboxylase (ODC)-PEST (proline-glutamate-serine-threonine)-rich protein (2). This GFP variant has a lower half-life (5.5 h) than native GFP in permanently transfected cells. When a genotoxic challenge was introduced (e.g. with a pulse of 140J/m2 UVC radiation or single dose of 100mM cyclohexamide), the % fraction of fluorescent cells containing the destabilised GFP-OCD-PEST dropped by 50% over 5h, and thereby recovered as the transcriptional block was removed by subsequent DNA repair over the next 3h.  By comparison, fluorescence from cells transfected with co...