Biomap analysis

Abstract

The involvement of an expression product in a cell in a pathway is determined by genetically modifying the cell, incubating the cell with predetermined factors in induce a physiological state and measuring parameters affected by the pathway. Changes in the levels of the parameters as a result of the presence of the expressed product indicate that the expression product is involved with the pathway.

1 . A method for characterization of a genetic agent according to its effect on cellular signaling pathways, the method comprising: 
 expressing said genetic agent in a cell, wherein said cell is present in a cell culture assay combination, wherein said cell culture assay combination comprises cells and at least one factor sufficient to provide a physiological state of interest involving at least two pathways;    recording changes in at least two different cellular parameter readouts;    deriving a biomap dataset from said changes in parameter readouts wherein said biomap comprises data normalized to control data on the same cell type under control conditions;    comparing said biomap dataset to a reference biomap dataset to determine the presence of variation, wherein the presence of variation indicates a difference in the effect of the genetic agent on a cellular signaling pathway.    
     
     
         2 . The method according to  claim 1 , wherein said assay combination comprises at least two factors.  
     
     
         3 . The method according to  claim 1 , wherein said gene is introduced into said cell by transfection.  
     
     
         4 . The method according to  claim 1 , wherein at least four parameters are measured.  
     
     
         5 . The method according to  claim 1 , further comprising the step of expressing said genetic agent in a panel of cell culture assay combinations.  
     
     
         6 . The method according to  claim 5 , wherein said assay combinations in said panel vary in the factors or cells that are present.  
     
     
         7 . The method according to  claim 1 , wherein said cells are endothelial cells.  
     
     
         8 . The method according to  claim 7 , wherein said endothelial cells are primary cells.  
     
     
         9 . The method according to  claim 8 , wherein said cells are primary HUVEC cells.  
     
     
         10 . The method according to  claim 7 , wherein said pathway is a pathway associated with inflammation.  
     
     
         11 . The method according to  claim 1 , wherein said cell is a leukocyte, cancer cell, or mast cell.  
     
     
         12 . The method according to  claim 10 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.  
     
     
         13 . The method according to  claim 1 , wherein said expression product is constitutively active.  
     
     
         14 . The method according to  claim 1 , wherein said genetic agent encodes an antisense nucleic acid.  
     
     
         15 . A method for determining whether a gene expression product influences at least one pathway in a cell in a cell culture, wherein said cell culture comprises an assay combination with at least two factors sufficient to provide a physiological state of interest involving at least two pathways in said cell in said cell culture, said pathways being indicated by levels of selected parameters, and said cell is genetically modified to overproduce as compared to a cell in the basal state, said method comprising: 
 incubating said genetically modified cell in said culture for sufficient time for expression of said gene and growth of said cell;    incubating said basal state cell in said culture for a time analogous to said incubating of said genetically modified cell; and    measuring the levels of said parameters for said genetically modified cell and said basal level cell;    whereby changes in said levels from said genetically modified cell from said basal level cell is indicative of the influence of said expression product on said at least one pathway.    
     
     
         16 . The method according to  claim 15 , wherein said genetically modified cell and said basal level cell are endothelial cells, leukocytes, cancer cells or mast cells.  
     
     
         17 . The method according to  claim 15 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.  
     
     
         18 . The method according to  claim 15 , wherein said at least one pathway includes at least one of NFκB and Jak/STAT.  
     
     
         19 . A method for determining whether a gene expression product influences at least one pathway in a cell in a cell culture, wherein a pathway includes at least one of NFκB and Jak/STAT, wherein said cell culture comprises an assay combination with at least two factors sufficient to provide a physiological state of interest involving at least two pathways in said cell in said cell culture, wherein said factors include at least one of IL-1, IFN-gamma, and TNF-alpha, said pathways being indicated by levels of selected; parameters, and said cell is genetically modified to overproduce as compared to the basal state the expression product of said gene, said method comprising: 
 incubating said genetically modified cell in said culture for sufficient time for expression of said gene and growth of said cell;    incubating said basal level cell in said culture for about the same time as said genetically modified cell is incubated; and    measuring the levels of said parameters for both said genetically modified cell and said basal level cell;    whereby changes in said levels of said genetically modified cell from said basal level cell is indicative of the influence of said expression product on said at least one pathway.    
     
     
         20 . The method according to  claim 19 , wherein said at least one pathway is involved with the inflammatory state.  
     
     
         21 . The method according to  claim 19 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.  
     
     
         22 . The method according to  claim 19 , wherein said changes in said levels is compared to at least one BioMAP of levels of parameters for a selected state.