Production of functional proteins: balance of shear stress and gravity

Abstract

The present invention provides for a method of culturing cells and inducing the expression of at least one gene in the cell culture. The method provides for contacting the cell with a transcription factor decoy oligonucleotide sequence comprising a nucleotide sequence encoding a shear stress response element.

1 - 26 . (canceled)  
     
     
         27 . A shear stress response element (SSRE) transcription factor decoy oligonucleotide comprising the nucleotide sequence selected from the group consisting of GAGACC and GGTCTC.  
     
     
         28 . The nucleotide of  claim 27 , wherein said oligonucleotide comprises a terminal phosphothioate moiety and a phosphodiester backbone.  
     
     
         29 . The nucleotide of  claim 27 , wherein said oligonucleotide is a contiguous single-stranded oligonucleotide comprising: i) a sequence selected from GAGACC and GGTCTC; and ii) a sequence compimentary to (i).  
     
     
         30 . The nucleotide of  claim 27 , wherein said oligonucleotide consists of SEQ ID NO: 1.  
     
     
         31 . The nucleotide of  claim 27 , wherein said oligonucleotide passes cell membranes and accumulates in the nuclear compartment of a cultured cell.  
     
     
         32 . The nucleotide of  claim 31  wherein said cultured cell is grown in two dimensional culture.  
     
     
         33 . The nucleotide of  claim 31  wherein said cultured cell is selected from the group consisting of an epithelial cell and an endothelial cell.  
     
     
         34 . The nucleotide of  claim 31  wherein said cultured cell is selected from the group consisting of renal cortical cell, renal fibroblast cell, hepatocyte, pancreatic islet, renal interstitial cell, parathyroid cell, thyroid cell, pituitary cell, ovarian cell and testicular cell.  
     
     
         35 . A method of producing biomolecules comprising the steps of: 
 culturing at least one cell;    contacting said cell with an SSRE transcription factor decoy oligonucleotide comprising a shear stress response element sequence selected from the group consisting of GAGACC and GGTCTC; and    isolating biomolecules from the cell culture.    
     
     
         36 . The method of  claim 35 , wherein said oligonucleotide comprises a terminal phosphothiorate phosphothioate moiety and a phosphodiester backbone.  
     
     
         37 . The method of  claim 36 , wherein said oligonucleotide passes cell membranes and accumulates in the nuclear compartment of said cell.  
     
     
         38 . The method of  claim 35 , wherein said cultured cell is selected from the group consisting of an epithelial cell and an endothelial cell.  
     
     
         39 . The method of  claim 35 , wherein said cultured cell is selected from the group consisting of renal cortical cell, renal fibroblast cell, hepatocyte, pancreatic islet, renal interstitial cell, parathyroid cell, thyroid cell, pituitary cell, ovarian cell and testicular cell.  
     
     
         40 . The method of  claim 35 , wherein said cultured cell is grown in two dimensional culture.  
     
     
         41 . The method of  claim 35 , wherein said cultured cell is grown in a rotating wall vessel.  
     
     
         42 . The method of  claim 35 , wherein the biomolecule is selected from the group consisting of megalin, cubulin, erythropoietin, 1-α-hydroxylase, and 1,25-dihydroxy-vitamin D3.  
     
     
         43 . A method of producing renal tubular epithelial cells from a cultured cell, comprising the steps of: 
 culturing at least one cell;    contacting said cell with an SSRE transcription factor decoy oligonucleotide comprising a shear stress response element sequence selected from the group consisting of GAGACC and GGTCTC; and    isolating differentiated renal tubular epithelial cells.    
     
     
         44 . The method of  claim 43 , wherein said cultured cell is selected from the group consisting of an epithelial cell and an endothelial cell.  
     
     
         45 . The method of  claim 43 , wherein said cultured cell is grown in a rotating wall vessel.  
     
     
         46 . The method of  claim 43 , wherein the renal tubular epithelial cell produces biomolecules selected from the group consisting of megalin, cubulin, erythropoietin, 1-α-hydroxylase, and 1,25-dihydroxy-vitamin D3.