Substances causing differentiation

Abstract

A DNA construct is described which contains a fusion gene under the control of a promoter. The fusion gene comprises at least one resistance gene and at least one reporter gene and is slightly toxic to a host cell transfected with that DNA construct. That DNA construct can be encoded on a plasmid or a virus. Further, a method is described for using the DNA construct to identify substances that may cause a differentiation in eukaryotic cells.

1 . A DNA construct, comprising a fusion gene under the control of a promoter, wherein said fusion gene comprises a resistance gene and a reporter gene and is slightly toxic to a host cell transfected with said DNA construct.  
   
   
       2 . The DNA construct of  claim 1 , wherein the promoter is selected from the group consisting of: a CMV promoter, an RSV promoter, cellular promoters of tumor suppressor genes and promoters of differentiation genes.  
   
   
       3 . The DNA construct of  claim 2 , wherein the reporter gene is selected from the group consisting of: GFP, LacZ and luciferase.  
   
   
       4 . The DNA construct of  claim 3 , wherein the resistance gene is selected from the group consisting of: a hygromycin gene, a neomycin gene and a puromycin gene.  
   
   
       5 . The DNA construct of  claim 2 , wherein the resistance gene is selected from the group consisting of a hygromycin gene, neomycin gene and puromycin gene.  
   
   
       6 . The DNA construct of  claim 1 , wherein the reporter gene is selected from the group consisting of: GFP, LacZ and luciferase.  
   
   
       7 . The DNA construct of  claim 6 , wherein the resistance gene is selected from the group consisting of: a hygromycin gene, a neomycin gene and a puromycin gene.  
   
   
       8 . The DNA construct of  claim 1 , wherein the resistance gene is selected from the group consisting of: a hygromycin gene, neomycin gene and puromycin gene.  
   
   
       9 . The DNA construct of  claim 1 , wherein the fusion gene comprises a gene coding for a polypeptide capable of developing an action toxic to the host cell.  
   
   
       10 . A plasmid, comprising a DNA construct of  claim 1 .  
   
   
       11 . A plasmid, comprising a DNA construct of  claim 2 .  
   
   
       12 . A plasmid, comprising a DNA construct of  claim 6 .  
   
   
       13 . A plasmid, comprising a DNA construct of  claim 8 .  
   
   
       14 . A plasmid, comprising a DNA construct of  claim 9 .  
   
   
       15 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 1  or codes therefor.  
   
   
       16 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 2  or codes therefor.  
   
   
       17 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 6  or codes therefor.  
   
   
       18 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 8  or codes therefor.  
   
   
       19 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 9  or codes therefor.  
   
   
       20 . A virus, comprising an expression cassette which comprises the DNA construct of  claim 10  or codes therefor.  
   
   
       21 . A virus, comprising an expression cassette which comprises the DNA construct od  claim 14  or codes therefor.  
   
   
       22 . A eukaryotic cell, comprising the plasmid of  claim 10 .  
   
   
       23 . The eukariotic cell of  claim 22 , wherein said cell is a human cell.  
   
   
       24 . The eukariotic cell of  claim 22 , wherein said cell is from the cell line U87-HGFP which was deposited in accordance with the Budapest Treaty at the DSMZ in Brunswick, Germany, under deposition number DSMZ ACC 2473 on Nov. 9, 2000.  
   
   
       25 . A eukaryotic cell, comprising the virus of  claim 15 .  
   
   
       26 . The eukaryotic cell of  claim 25 , wherein said cell is a human cell.  
   
   
       27 . The eukaryotic cell of  claim 25 , wherein said cell is from the cell line U87-HGFP which was deposited in accordance with the Budapest Treaty at the DSMZ in Brunswick, Germany, under deposition number DSMZ ACC 2473 on Nov. 9, 2000.  
   
   
       28 . A method for identifying a compound capable of causing differentiation in eukaryotic cells, comprising: 
 a) incubating cells of  claim 22  in a culture medium containing a selection substance corresponding to the resistance gene;    b) inoculating a culture medium lacking said selection substance with the incubated cells from step a);    c) incubating the culture medium inoculated with the cells from step b) for about 5 hours to about 100 hours;    d) checking the incubated cells from step c) for expression of the reporter gene;    e) adding said compound to said culture medium of the cells from step c);    f) incubating said culture medium with the cells and said compound for about 1 day to about 5 days;    g) checking the incubated cells from step f) for expression of the reporter gene compared to cells from step d); and    e) identifying the compound that causes the increase in the expression of said reporter gene as the compound that causes differentiation in eukaryotic cells.    
   
   
       29 . The method of  claim 28 , wherein said incubating of the culture medium inoculated with the cells in step c) is for about 24 hours.  
   
   
       30 . The method of  claim 28 , wherein said incubating of said culture medium with the cells and said compound in step f) is for about 2 days.  
   
   
       31 . A method for identifying a compound capable of causing differentiation in eukaryotic cells, comprising: 
 a) incubating cells of  claim 25  in a culture medium containing a selection substance corresponding to the resistance gene;    b) inoculating a culture medium lacking said selection substance with the incubated cells from step a);    c) incubating the culture medium inoculated with the cells from step b) for about 5 hours to about 100 hours;    d) checking the incubated cells from step c) for expression of the reporter gene;    e) adding said compound to said culture medium of the cells from step c);    f) incubating said culture medium with the cells and said compound for about 1 day to about 5 days;    g) checking the incubated cells from step f) for expression of the reporter gene compared to cells from step d); and    e) identifying the compound that causes the increase in the expression of said reporter gene as the compound that causes differentiation in eukaryotic cells.    
   
   
       32 . The method of  claim 31 , wherein said incubating of the culture medium inoculated with the cells in step c) is for about 24 hours.  
   
   
       33 . The method of  claim 31 , wherein said incubating of said culture medium with the cells and said compound in step f) is for about 2 days.  
   
   
       34 . A method for preparing a pharmaceutical composition, comprising the steps of the method of  claim 28  and further comprising mixing the identified compund with a pharmaceutically acceptable carrier.  
   
   
       35 . A method for preparing a pharmaceutical composition, comprising the steps of the method of  claim 31  and further comprising mixing the identified compound with a pharmaceutically acceptable carrier.