Assays for mutagenesis detection
The disclosure provides methods, systems, and kits for assaying an agent for mutagenic properties. The methods systems and kits utilize a DEL selectable marker and a colorimetric detection systems. Also included are methods systems and kits that utilize a DEL selectable marker and a regent that detects mitochondrial activity.
1. A method for characterizing an agent, comprising:
treating a culture of eukaryotic cells comprising a DEL selection marker with an agent (“treated portion”) or without an agent (“untreated portion”);
measuring the mitochondrial activity of the treated portion of the cell culture in the presence of a suitable selection medium;
measuring the mitochondrial activity of the untreated portion of the cell culture in the presence of said selection medium; and
quantitating the change in mitochondrial activity and DEL induction between the treated portion and the untreated portions of the cell culture, wherein the change in mitochondrial activity and DEL induction indicates in a simultaneous manner both the cytotoxicity and mutagenicity of the agent;
wherein the eukaryotic cells comprise a construct having an auxotrophic gene disrupted by a colorigenic enzyme gene, and the colorigenic enzyme gene is disrupted by a polynucleotide encoding a selectable marker.
2. The method of claim 1 , wherein the mitochondrial activity is measured by detecting the reduction of MTT or MTS.
3. The method of claim 1 , wherein the cell culture comprises a cell selected from the group consisting of a mammalian lymphoid cell; a human lymphoblastoid cell; and a yeast cell.
4. A method of claim 3 , wherein the cell culture comprises Saccharomyces cerevisiae.
5. The method of claim 1 , wherein the measuring comprises detecting a colorimetric precipitate.
6. The method of claim 1 , wherein the method is performed in a multi-well plate.
7. The method of claim 1 , wherein the DEL selection marker is a metabolic enzyme or pathway.
8. The method of claim 1 , wherein the DEL selection marker is an anti-microbial resistance gene.
9. The method of claim 1 , wherein the DEL selection marker is the his gene.
10. The method of claim 1 , wherein the auxotrophic gene comprises a His3 gene and the selectable marker comprises a Ura3 gene.
11. The method of claim 1 , wherein the construct comprises a plasmid.
12. The method of claim 1 , wherein the colorigenic enzyme gene comprises LacZ.
13. The method of claim 1 , wherein the construct comprises the nucleic acid sequence set forth in SEQ ID NO: 1.
14. A method of screening an agent of mutagenic potential, comprising:
providing a cell culture comprising a plurality of micro-titer wells each well comprising a cell, each cell having a DEL selectable marker;
culturing a fraction of the cells with medium comprising an agent to be tested for mutagenicity and culturing a fraction of the cells with control medium;
washing the cells;
culturing the cells with an agent that measures mitochondrial activity; and
measuring a detectable signal indicative of mitochondrial activity;
wherein the cells are eukaryotic cells that comprise a construct having an auxotrophic gene disrupted by a colorigenic enzyme gene, and the colorigenic enzyme gene is disrupted by a polynucleotide encoding a selectable marker.
15. The method of claim 14 , wherein the cell is a yeast cell.
16. The method of claim 14 , wherein the agent is MTT or MTS.
17. The method of claim 14 , wherein the detectable signal is a colorimetric signal and the measuring is by absorbance.
18. The method of claim 14 , wherein the micro-titer wells are in a 96 or 384 well format.