Method for using division arrested cells in screening assays

Abstract

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 if a substance of interest has an effect on a G-protein coupled receptor (GPCR) signal in a division arrested mammalian cell, the method comprising:
 (a) contacting a division arrested mammalian cell with the substance of interest and measuring the GPCR signal in the cell, wherein the cell has been transformed or transfected with a nucleic acid encoding the GPCR, and   (b) determining whether the substance of interest has an effect on the GPCR signal in the mammalian cell by   identifying a comparative difference in the signal when the mammalian cell has and has not been contacted with the substance of interest.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the G-protein coupled receptor is selected from the group consisting of:
 (a) a serotonin receptor;   (b) a muscaranic acetylcholine receptor; and   (c) a β2 adrenergic receptor.   
     
     
         5 . The method of  claim 1 , wherein the GPCR signal measured in the mammalian cell results from Ca 2+  mobilization. 
     
     
         6 . The method of  claim 1 , wherein the GPCR signal measured in the mammalian cell is determined by measuring the activity of a reporter. 
     
     
         7 . The method of  claim 6 , wherein the reporter is selected from the group consisting of secreted alkaline phosphatase, luciferase, and green fluorescent protein. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the mammalian cell is a cell selected from the group consisting of:
 (a) a Chinese hamster ovary cell;   (b) a NIH3T3 cell;   (c) a HEK293 cell; and   (d) a M1WT3 cell.   
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the substance of interest has efficacy as a GPCR agonist. 
     
     
         12 . The method of  claim 1 , wherein the substance of interest has efficacy as a GPCR antagonist. 
     
     
         13 . The method of  claim 1 , wherein the division arrested mammalian cell has been frozen and thawed before contact with the substance of interest. 
     
     
         14 . The method of  claim 1 , wherein the mammalian cell was irradiated to arrest cellular division. 
     
     
         15 . A composition comprising a population of mammalian cells, wherein the population is substantially uniformly division arrested and wherein the cells of the population comprise:
 (i) a transformed or transfected nucleic acid encoding a G-protein coupled receptor (GPCR);   (ii) the encoded GPCR; and   (iii) a substance of interest, wherein the substance of interest is capable of having an effect on the activity of the GPCR.   
     
     
         16 . The composition of  claim 15 , wherein the cells of the population are stably transformed or transfected with the nucleic acid. 
     
     
         17 . The composition of  claim 15 , wherein the GPCR is selected from the group consisting of:
 (a) a serotonin receptor;   (b) a muscaranic acetylcholine receptor; and   (c) a β2 adrenergic receptor.   
     
     
         18 . The composition of  claim 15 , wherein the cells are selected from the group consisting of:
 (a) Chinese hamster ovary cells (CHO);   (b) NIH3T3 cells;   (c) HEK293 cells; and   (d) M1WT3 cells.   
     
     
         19 . The composition of  claim 15 , wherein the substance has efficacy as a GPCR agonist. 
     
     
         20 . The composition of  claim 15 , wherein the substance has efficacy as a GPCR antagonist. 
     
     
         21 . The composition of  claim 15 , wherein the population of cells has been frozen and thawed before contact with the substance of interest. 
     
     
         22 . The composition of  claim 15 , wherein the cells were irradiated to arrest cellular division. 
     
     
         23 . A composition comprising a population of mammalian cells, wherein the population is substantially uniformly division arrested, wherein the cells of the population are transformed or transfected with a nucleic acid encoding a G-protein coupled receptor (GPCR) and express the encoded GPCR; and
 wherein the composition is frozen and the cells of the population remain in division arrest after the composition is thawed.   
     
     
         24 . The composition of  claim 23 , wherein the cells of the population are stably transformed or transfected with the nucleic acid. 
     
     
         25 . The composition of  claim 23 , wherein the GPCR is selected from the group consisting of:
 (a) a serotonin receptor;   (b) a muscaranic acetylcholine receptor; and   (c) a β2 adrenergic receptor.   
     
     
         26 . The composition of  claim 23 , wherein the cells are selected from the group consisting of:
 (a) Chinese hamster ovary cells (CHO);   (b) NIH3T3 cells;   (c) HEK293 cells; and   (d) M1WT3 cells.   
     
     
         27 . The composition of  claim 23 , wherein the cells were irradiated to arrest cellular division.