Levels and/or Sustainability of DNA-based Gene Expression

Abstract

The invention encompasses methods for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising: (a) modifying the target nucleic acid to introduce or to comprise signals that limit or constrain the positions of nucleosome cores, and (b) introducing the modified target nucleic acid into the eukaryotic cell, wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

1 - 62 . (canceled) 
     
     
         63 . A synthetic deoxyribonucleic acid (DNA) encoding a protein, the synthetic DNA being double stranded with one strand comprising a coding sequence encoding the protein with the coding sequence being interrupted by at least one synthetic intron, wherein the synthetic intron is at least 20 bases length and comprises at least two consecutive inserts that are 10 to 11 bases in length, wherein the inserts are on the same nucleic acid strand as the strand encoding the GFP and comprise a sequence of 2-5 consecutive bases of adenine (A) and/or thymine (T). 
     
     
         64 . The synthetic DNA of  claim 63 , wherein the synthetic intron comprises at least three consecutive inserts of 10 or 11 bases in length. 
     
     
         65 . The synthetic DNA of  claim 64 , wherein at least one of the synthetic introns is 90 bases in length or less. 
     
     
         66 . The synthetic DNA of  claim 65 , wherein the coding sequence that encodes the protein is interrupted by 2, 3, 4, 5, 6, 7, 8, 9 or 10 synthetic introns. 
     
     
         67 . The synthetic DNA of  claim 63 , wherein the synthetic DNA is contained in an expression vector. 
     
     
         68 . The synthetic DNA of  claim 67 , wherein the expression vector comprises a constitutive promoter. 
     
     
         69 . The synthetic DNA of  claim 67 , wherein the expression vector comprises an inducible promoter. 
     
     
         70 . The synthetic DNA of  claim 67 , wherein the expression vector is contained within a host cell. 
     
     
         71 . The synthetic DNA of  claim 70 , wherein the host cell is a eukaryotic cell. 
     
     
         72 . A method of expressing a protein in a host cell, the method comprising
 a) introducing into the host cell a synthetic deoxyribonucleic acid (DNA) encoding the protein, the synthetic DNA being double stranded with one strand comprising a coding sequence encoding the protein with the coding sequence being interrupted by at least one synthetic intron, wherein the synthetic intron is at least 20 bases length and comprises at least two consecutive inserts that are 10 to 11 bases in length, wherein the inserts are on the same nucleic acid strand as the strand encoding the GFP and comprise a sequence of 2-5 consecutive bases of adenine (A) and/or thymine (T), and   b) providing conditions to the host cell that are suitable for protein expression.   
     
     
         73 . The method of  claim 72 , wherein the synthetic intron of the introduced synthetic DNA comprises at least three consecutive inserts of 10 or 11 bases in length. 
     
     
         74 . The method of  claim 73 , wherein at least one of the synthetic introns in the introduced synthetic DNA is 90 bases in length or less. 
     
     
         75 . The method of  claim 74 , wherein the coding sequence that encodes the protein in the introduced synthetic DNA is interrupted 2, 3, 4, 5, 6, 7, 8, 9 or 10 synthetic introns. 
     
     
         76 . The method of  claim 72 , wherein the introduced synthetic DNA is contained in an expression vector. 
     
     
         77 . The method of  claim 76 , wherein the expression vector comprises a constitutive promoter. 
     
     
         78 . The method of  claim 76 , wherein the expression vector comprises an inducible promoter. 
     
     
         79 . The method of  claim 76 , wherein the expression vector is contained within a host cell. 
     
     
         80 . The method of  claim 79 , wherein the host cell is in culture and providing the conditions to the host cell that are suitable for expression of the protein comprises culturing the host cell in culture conditions suitable for protein expression in culture. 
     
     
         81 . The method of  claim 79 , wherein the host cell is a eukaryotic cell. 
     
     
         82 . The method of  claim 81 , wherein the host cell is plant cell or an animal cell.