Method for using division arrested cells in screening assays
View Patent ↗Division arrested cells are used in screening assays to determine the effect of a substance of interest on the cells. The division arrested cells can be used in drug screening assays, signal transduction assays, and are especially useful in large scale, high throughput assays.
1. A method for determining is a substance of interest interacts with a G protein coupled receptor (GPCR), comprising:
(i) contacting said substance of interest with a cell which expresses said G protein coupled receptor, wherein said cell has had its division arrested, so the either its mitotic or meiotic cycle has been stopped, and cellular division can no longer take place,
(ii) contacting said substance of interest with a cell which does not express said G protein coupled receptor, wherein said cell has had its division arrested, so that either its mitotic or meiotic cycle has been stopped, and cellular division can no longer take place, and
(iii) comparing any change in the cell of (i) to any change in the cell of (ii), wherein a difference therebetween is indicative of modulation of said G coupled protein receptor with said substance of interest.
2. A method for determining if a substance of interest interacts with a G protein coupled receptor (GPCR), comprising:
(i) contacting said substance of interest with a cell which expresses said G protein coupled receptor, wherein said cell has had its division arrested, so the either its mitotic or meiotic cycle has been stopped, and cellular divisional can no longer take place,
(ii) contacting said substance of interest with a cell which expresses said G protein coupled receptor, wherein said cell has had its division arrested so that either its mitotic or meiotic cycle has been stopped, and cellular division can no longer take place, wherein said cell has been treated with a G protein coupled receptor antagonist prior to contact with said substance of interest, and
(iii) comparing any change in the cell of (i) to that of (ii) wherein a difference therebetween is indicated of modulation of said G coupled protein receptor by said substance of interest.
3. The method of claim 1 or 2 , wherein said cell is a eukaryotic cell.
4. The method of claim 1 or 2 , wherein said cell is a prokaryotic cell.
5. The method of claim 1 or 2 , wherein division of said cell has been arrested during mitosis.
6. The method of claim 1 or 2 , wherein division of said cell has been arrested during meiosis.
7. The method of claim 1 or 2 , wherein said substance of interest is a protein and said cell has been transformed or transfected with a nucleic acid molecule which expresses said GPCR.
8. The method of claim 7 , wherein said cell has been transformed or transfected with a recombinant virus vector which expresses said GPCR.
9. The method of claim 1 or 2 , wherein said cell has been transformed or transfected with RNA which encodes said GPCR.
10. The method of claim 1 or 2 , wherein said cell has been transduced with a protein, peptide, or protein containing molecule.
11. The method of claim 1 or 2 , wherein said cell is an NIH3T3 cell.
12. The method of claim 1 or 2 , said method comprises measuring induction of Ca2+ immobilization.
13. The method of claim 1 or 2 , wherein said cell has been transformed or transfected with a nucleic acid molecule which encodes said 5HT2c.
14. The method of claim 1 or 2 , wherein said cell has been transfected or transformed with a nucleic acid molecule which expresses said G-protein coupled receptor.
15. The method of claim 1 or 2 , wherein said cells express ion channels.
16. The method of claim 14 , wherein said G-protein coupled receptor is muscaranic acetylcholine receptor.
17. The method of claim 1 or 2 , wherein said cell is an HEK293 cell.
18. The method of claim 1 or 2 , wherein said cell expresses a ÿ2 adrenergic receptor.
19. The method of claim 1 or 2 , wherein said cell has been transformed or transfected with a nucleic acid molecule which encodes a reporter molecule, wherein said reporter molecule gives a detectable signal upon modulation of said GPCR by said substance of interest.
20. The method of claim 19 , wherein said reporter molecule is expressed due to activation of a molecule expressed in said cell, whose activation is caused by interaction of said GPCR and said substance of interest.
21. The method of claim 19 , wherein said reporter molecule is luciferase, or green fluorescent protein.
22. The method of claim 1 or 2 , said method comprising a competitive assay wherein said substance of interest is admixed with a compound known to effect said GPCR, and determining any change in the known effect as a determination that said substance of interest effects said GPCR.