METHOD FOR DETERMINING GENOTOXICITY
First Claim
Patent Images
1. A cell comprising:
- a) a first reporter gene construct comprising a nucleic acid molecule encoding a fluorescent protein fused to a centromere protein said nucleic acid sequence operably linked to and under the control of a promoter that is constitutively active; and
b) a second reporter gene construct comprising a nucleic acid molecule comprising one or more expression control sequences operably linked to a sequence encoding a nitroreductase (NTR) enzyme.
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Abstract
Disclosed are compositions and methods which provide an integrated approach to genotoxicity assessment procedures by employing a genetically engineered cell line to report on a number of key indicators of genotoxicity effects and mechanisms. Imaging and analysis of cells exposed to test agents allows automated analysis using high content cellular screening to identify cytostatic and/or cytotoxic activity, to quantify micronuclei formation, to discriminate aneugenic and clastogenic micronuclei and to detect DNA mutation and repair.
12 Citations
33 Claims
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1. A cell comprising:
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a) a first reporter gene construct comprising a nucleic acid molecule encoding a fluorescent protein fused to a centromere protein said nucleic acid sequence operably linked to and under the control of a promoter that is constitutively active; and b) a second reporter gene construct comprising a nucleic acid molecule comprising one or more expression control sequences operably linked to a sequence encoding a nitroreductase (NTR) enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A cell line comprising at least one cell engineered to:
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a) constitutively express a fluorescent protein in fusion with a centromere protein; and b) inducibly express a nitroreductase (NTR) enzyme reporter gene under the control of a DNA damage induced promoter. - View Dependent Claims (10, 11, 12, 13, 14)
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15. A process for producing a cell line including at least one cell comprising first and second reporter gene constructs capable of being expressed, which process comprises:
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a) transfecting one or more cells with a first and a second recombinant expression vector, wherein; i) said first recombinant expression vector comprises a first reporter gene construct comprising a nucleic acid molecule encoding a fluorescent protein fused to a centromere protein said nucleic acid sequence operably linked to and under the control of a promoter that is constitutively active; and ii) said second recombinant expression vector comprises a second reporter gene construct comprising a nucleic acid molecule comprising one or more expression control sequences operably linked to a sequence encoding a nitroreductase (NTR) enzyme; and b) selecting transfected cells which express at least one and optionally both first and second reporter genes. - View Dependent Claims (16)
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17. A method for determining the effect of a test agent on a phenotypic property of a cell, which method comprises:
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a) providing cells comprising; i) a first reporter gene construct comprising a nucleic acid molecule encoding a fluorescent protein capable of emitting fluorescent light of a first wavelength (λ
1) and being fused to a centromere protein said nucleic acid sequence operably linked to and under the control of a promoter that is constitutively active; andii) a second reporter gene construct comprising a nucleic acid molecule comprising one or more expression control sequences operably linked to a sequence encoding a nitroreductase (NTR) enzyme; and wherein said cells are marked with a DNA staining dye that emits fluorescent light of a second wavelength (λ
2) when bound to DNA;b) contacting a population of said cells with; iii) a test agent or a control; and iv) a substrate for nitroreductase wherein said substrate is capable of emitting fluorescent light of a third wavelength (λ
3);under conditions permitting expression of said first and second reporter gene constructs; and c) measuring the fluorescence intensity at different wavelengths of emitted light in said cell population; wherein a difference in fluorescence intensity in cells treated with said test agent compared with cells treated with said control is indicative of the effect of said test agent on the phenotypic property of said cell. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A diagnostic kit for identifying and characterising a toxic agent, said kit comprising:
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a) at least one cell comprising; i) a first reporter gene construct comprising a nucleic acid molecule encoding a fluorescent protein fused to a centromere protein said nucleic acid sequence operably linked to and under the control of a promoter that is constitutively active; and ii) a second reporter gene construct comprising a nucleic acid molecule comprising one or more expression control sequences operably linked to a sequence encoding a nitroreductase (NTR) enzyme; and b) a nitroreductase (NTR) enzyme substrate.
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Specification