Levels and/or sustainability of DNA-based gene expression
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. A DNA molecule encoding green fluorescent protein (GFP), the DNA molecule being double stranded with one strand comprising a coding sequence encoding the GFP with the coding sequence being interrupted by at least one synthetic intron, wherein the at least one synthetic intron is at least 20 bases in length and comprises at least two consecutive inserts that are 10 to 11 bases in length, wherein the inserts are on the same DNA strand as the strand encoding the GFP and comprise a sequence of 2-5 consecutive bases of adenine (A) and/or thymine (T).
2. The DNA molecule of claim 1 , wherein at least one of the at least one synthetic introns comprises at least three consecutive inserts of 10 or 11 bases in length.
3. The DNA molecule of claim 2 , wherein at least one of the at least one synthetic introns is 90 bases in length or less.
4. The DNA molecule of claim 3 , wherein the coding sequence that encodes the GFP is interrupted by 2, 3, 4, 5, 6, 7, 8, 9 or 10 synthetic introns.
5. The DNA molecule of claim 1 , wherein at least one of the at least one synthetic introns comprises the nucleotide sequence of SEQ ID NO: 8.
6. The DNA molecule of claim 5 , wherein the DNA molecule comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:2.
7. The DNA molecule of claim 6 , wherein the DNA molecule comprises the nucleotide sequence of SEQ ID NO:2.
8. The DNA molecule of claim 1 , wherein the DNA molecule is contained in an expression vector.
9. The DNA molecule of claim 8 , wherein the expression vector is adenovirus vector.
10. The DNA molecule of claim 8 , wherein the expression vector comprises a constitutive promoter.
11. The DNA molecule of claim 8 , wherein the expression vector comprises an inducible promoter.
12. The DNA molecule of claim 8 , wherein the expression vector is contained within a eukaryotic cell.
13. The DNA molecule of claim 8 , wherein the expression vector comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO:12.
14. The DNA molecule of claim 13 , wherein the expression vector comprises the nucleic acid sequence of SEQ ID NO:12.
15. A method of expressing green fluorescent protein (GFP) in a eukaryotic cell, the method comprising:
a) introducing into the eukaryotic cell a DNA molecule encoding green fluorescent protein (GFP), the DNA molecule being double stranded with one strand comprising a coding sequence encoding the GFP with the coding sequence being interrupted by least one synthetic intron, wherein the at least one synthetic intron is at least 20 bases in length and comprises at least two consecutive inserts that are 10 to 11 bases in length, wherein the inserts are on the same DNA molecule 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 eukaryotic cell that are suitable for protein expression, wherein the eukaryotic cell expresses the GFP.
16. The method of claim 15 , wherein the GFP is expressed longer than standard humanized GFP.
17. The method of claim 15 , wherein the at least one synthetic intron of the introduced DNA molecule comprises at least three consecutive inserts of 10 or 11 bases in length.
18. The method of claim 15 , wherein the at least one synthetic intron in the introduced DNA molecule is 90 bases in length or less.
19. The method of claim 15 , wherein the coding sequence that encodes the GFP in the introduced DNA molecule is interrupted by 2, 3, 4, 5, 6, 7, 8, 9 or 10 synthetic introns.
20. The method of claim 15 , wherein at least one of the at least one synthetic introns of the introduced DNA molecule comprises the nucleotide sequence of SEQ ID NO: 8.
21. The method of claim 15 , wherein the introduced DNA molecule comprises a nucleic acid sequence at least 80% identical to nucleic acid sequence of SEQ ID NO:2.
22. The method of claim 21 , wherein the introduced DNA molecule comprises the nucleic acid sequence of SEQ ID NO:2.
23. The method of claim 15 , wherein the introduced DNA molecule is contained in an expression vector.
24. The method of claim 23 , wherein the expression vector comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO:12.
25. The method of claim 24 , wherein the expression vector comprises the nucleic acid sequence of SEQ ID NO:12.
26. The method of claim 24 , wherein the expression vector is an adenovirus vector.
27. The method of claim 26 , wherein the expression vector comprises a constitutive promoter.
28. The method of claim 23 , wherein the expression vector comprises an inducible promoter.