Expression vector for consistent cellular expression of the tet on repressor in multiple cell types

Abstract

The present invention provides a vector comprising Protein Translation Peptide Elongation Factor-1 α promoter and nucleic acids encoding reverse tetracycline controlled transactivator, wherein the expression of said transactivator is under the control of Protein Translation Peptide Elongation Factor-1 α promoter. In addition, the invention provides a method of generating a reverse tetracycline controlled transactivator expression system for inducible tetracycline regulated gene expression comprising: (a) isolation of a DNA fragment encoding the reverse tetracycline controlled transactivator by restriction enzyme digestion, (b) generation of Protein Translation Peptide Elongation Factor-1 α promoter vector, by restriction enzyme digestion, (c) directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 α promoter vector by ligation of 5′ EcoRi compatible restriction enzyme overhangs, (d directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 α promoter vector by Klenow fragment mediated blunt end generation of 3′ Bam HI end of DNA fragment encoding the reverse tetracycline controlled transactivator and 3′ XbaI end of Protein Translation Peptide Elongation Factor-1 α promoter vector and (e) blunt cloning of partially ligated fragment to produce Protein Translation Peptide Elongation Factor-I α promoter vector expressing reverse tetracycline controlled transactivator.

1 . A vector comprising: 
 a) a human Protein Translation Peptide Elongation Factor-1 α promoter;    b) a nucleic acid encoding a tetracycline controlled transactivator, wherein the expression of said transactivator is under the control of the promoter;    c) a tetracycline inducible operator binding element under the control of the nucleic acid encoding the transactivator, and    d) a gene of interest under the control of the promoter.    
     
     
         2 . The vector of  claim 1  , wherein the vector is a plasmid.  
     
     
         3 . The vector of  claim 1 , wherein the vector is as set forth in FIG. 1.  
     
     
         4 . An isolated cell comprising the vector of  claim 1 .  
     
     
         5 . The cell of  claim 4 , wherein the cell is from a cell line.  
     
     
         6 . The cell of  claim 5 , wherein the cell line is HeLa (human cervix), HO-1 (human melanoma), MCF-7(human breast), PC3 (human prostate) or DU-145 (human prostate).  
     
     
         7 . The cell of  claim 4 , which consistently expresses tetracycline repressor.  
     
     
         8 . A cell comprised of Protein Translation Peptide Elongation Factor-1 α promoter and nucleic acids encoding reverse tetracycline controlled transactivator, wherein the expression of said transactivator is under the control of Protein Translation Peptide Elongation Factor-1 α promoter.  
     
     
         9 . A non-human animal comprising the vector of  claim 1 .  
     
     
         10 . The animal of  claim 9 , wherein the animal is a mouse.  
     
     
         11 . A method of generating a reverse tetracycline controlled transactivator expression system for inducible tetracycline regulated gene expression comprising: 
 a) isolation of a DNA fragment encoding the reverse tetracycline controlled transactivator by restriction enzyme digestion.    b) generation of Protein Translation Peptide Elongation Factor-1 α promoter vector, by restriction enzyme digestion;    c) directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 α promoter vector by ligation of 5′ EcoRI compatible restriction enzyme overhangs;    d) directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 α promoter vector by Klenow fragment meiadte blunt end generation of 3′ Bam HI end of DNA fragment encoding the reverse tetracycline controlled transactivator and 3′ XbaI end of Protein Translation Peptide Elongation Factor-1 α promoter vector; and    e) blunt cloning of partially ligated fragment to produce Protein Translation Peptide Elongation Factor-1 α promoter vector expressing reverse tetracycline controlled transactivator.    
     
     
         12 . The method of  claim 11 , wherein the fragment of  11 ( a ) is an Eco RI-BAM HI fragment.  
     
     
         13 . The method of  claim 11 , wherein the mammalian expression vector of  11 ( b ) is pCDEF3.  
     
     
         14 . The method of  claim 11 , wherein the cloning of  11 ( a ) is at the 5′ Eco RI and 3′ BAM HI sites.  
     
     
         15 . The method of  claim 11 , wherein the ligation of  11 ( c ) is at the 5′ Eco RI site of pCDEF3.  
     
     
         16 . The method of  claim 11 , wherein the ligation of  11 ( d ) is at the 3′ XbaI site of pCDEF3.  
     
     
         17 . A vector generated by the method of  claim 11 .  
     
     
         18 . A method for screening for an anti-tumor drug which comprises administering to a transgenic non-human animal a drug wherein the animal inducibly expresses or represses expression of a gene of interest under regulation of tetracycline or deoxycycline and wherein the gene of interest is associated with cancer, and determining whether the animal develops a tumor thereby screening for an anti-tumor drug.  
     
     
         19 . A method for expressing a gene of interest which comprises contacting the cell of  claim 4  with an inducer of the tetracycline inducible operator binding element so as to cause the cell to express the gene of interest.  
     
     
         20 . The method of  claim 19 , wherein the inducer is tetracycline or deoxycycline.