Method for determining cell cycle position

Abstract

The invention provides a novel, non-destructive and dynamic process for determining the cell cycle position of living cells. The invention also provides DNA constructs, and cell lines containing such constructs, that exhibit activation and deactivation of a detectable reporter molecule in a cell cycle specific manner. The invention thus allows greater precision in determining cell cycle phase status than existing techniques and further provides a method for continuous monitoring of cell cycle progression in individual cells.

1 - 18 . (canceled)  
     
     
         19 . A method for determining the cell cycle position of a cell, said method comprising: 
 a) expressing in a cell a nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live-cell reporter molecule operably linked to and under the control of: 
 i) a cell cycle phase-specific expression control element, and  
 ii) a destruction control element;  
   wherein said reporter construct is expressed in a cell at a predetermined point in the cell cycle; and    b) determining the cell cycle position by monitoring signals emitted by the reporter molecule.    
     
     
         20 . A method of determining the effect of a test system on the cell cycle position of a cell, said method comprising: 
 a) expressing in said cell in the absence and in the presence of said test system a nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live-cell reporter molecule operably linked to and under the control of: 
 i) a cell cycle phase-specific expression control element, and  
 ii) a destruction control element;  
   wherein said reporter construct is expressed in a cell at a predetermined point in the cell cycle; and    b) determining the cell cycle position by monitoring signals emitted by the reporter molecule wherein a difference between the emitted signals measured in the absence and in the presence of said test system is indicative of the effect of the test system on the cell cycle position of the cell.    
     
     
         21 . A method of determining the effect of a test system on the cell cycle position of a cell, said method comprising: 
 a) expressing in said cell in the presence of said test system a nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live-cell reporter molecule operably linked to and under the control of: 
 i) a cell cycle phase-specific expression control element, and  
 ii) a destruction control element;  
   wherein said reporter construct is expressed in a cell at a predetermined point in the cell cycle; and    b) determining the cell cycle position by monitoring signals emitted by the reporter molecule,    c) comparing the emitted signal in the presence of the test system with a known value for the emitted signal in the absence of the test system;    wherein a difference between the emitted signal measured in the presence of the test system and said known value in the absence of the test system is indicative of the effect of the test system on the cell cycle position of the cell.    
     
     
         22 . A method of determining the effect of a test system on the cell cycle position of a cell, said method comprising: 
 a) providing cells containing a nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live-cell reporter molecule operably linked to and under the control of: 
 i) a cell cycle phase-specific expression control element, and  
 ii) a destruction control element;  
   wherein said reporter construct is expressed in a cell at a predetermined point in the cell cycle;    b) culturing first and second populations of said cells respectively in the presence and absence of a test system and under conditions permitting expression of the nucleic acid reporter construct; and    c) measuring the signals emitted by the reporter molecule in said first and second cell populations;    wherein a difference between the emitted signals measured in said first and second cell populations is indicative of the effect of said test system on the cell cycle position of said cell.    
     
     
         23 . A method of determining the effect of the cell cycle on the expression, translocation or sub-cellular distribution of a first detectable reporter which is known to vary in response to a test system, said method comprising: 
 a) expressing in said cell in the presence of said test system a second nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live-cell reporter molecule operably linked to and under the control of: 
 i) a cell cycle phase-specific expression control element, and  
 ii) a destruction control element;  
   wherein said reporter construct is expressed in a cell at a predetermined point in the cell cycle;    b) determining the cell cycle position by monitoring signals emitted by the second reporter molecule; and    c) monitoring the signals emitted by said first detectable reporter,    wherein the relationship between cell cycle position determined by step b) and the signal emitted by the first detectable reporter is indicative of whether or not the expression, translocation or sub-cellular distribution of the first detectable reporter is cell cycle dependent.    
     
     
         24 . (canceled)  
     
     
         25 . The method of  claim 20 , wherein said test system is an agent selected from a drug, nucleic acid, hormone, protein, or a peptide to which said cell is exposed.  
     
     
         26 . The method of  claim 20 , wherein said test system is an agent selected from a peptide or protein that is expressed in the cell under study.  
     
     
         27 - 29 . (canceled)