DNA vectors, transposons and transposases for eukaryotic genome modification
The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.
1. An isolated eukaryotic cell whose genome comprises a polynucleotide comprising transposon ends of a piggyBac-like transposon flanking a heterologous polynucleotide not associated with the transposon ends in nature, wherein each transposon end comprises 5′-CCYTTTBMCTGCCA-3′ (SEQ ID NO: 19) inverted in orientation in the two transposon ends, and wherein the identity of nucleotides of an ambiguity code can be selected independently in the two repeats.
2. The isolated eukaryotic cell of claim 1 , wherein one transposon end further comprises a sequence that is at least 90% identical to SEQ ID NO: 9 and the other transposon end further comprises a sequence that is at least 90% identical to SEQ ID NO: 17.
3. The isolated eukaryotic cell of claim 1 , wherein the polynucleotide further comprises a gene encoding a selectable marker.
4. The isolated eukaryotic cell of claim 3 , wherein the selectable marker is one of the following: glutamine synthase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a protein that binds to zeocin.
5. The isolated eukaryotic cell of claim 3 , wherein the gene encoding the selectable marker is operably linked to a promoter which is at least 95% identical to the sequence of SEQ ID NO: 942.
6. The isolated eukaryotic cell of claim 1 , wherein the heterologous polynucleotide further comprises a eukaryotic promoter.
7. The isolated eukaryotic cell of claim 6 , wherein the promoter is selected from one of the following: an EF1a promoter, a CMV promoter, an EEF2 promoter, an SV40 promoter, a PGK promoter, an actin promoter, an AOX promoter, a GAPDH promoter, an ILV5 promoter or an HSV-TK promoter.
8. The isolated eukaryotic cell of claim 6 , wherein the promoter is at least 95% identical to the sequence of SEQ ID NO: 896.
9. The isolated eukaryotic cell of claim 1 , wherein the heterologous polynucleotide further comprises an intron.
10. The isolated eukaryotic cell of claim 9 , wherein the intron is selected from one of the following: an EF1a intron, an EEF2 intron, a CMV intron, an actin intron, a GAPDH intron, an SV40 intron.
11. The isolated eukaryotic cell of claim 9 , wherein the intron is at least 95% identical to the sequence of SEQ ID NO: 975.
12. The isolated eukaryotic cell of claim 1 , wherein the polynucleotide further comprises a promoter and an intron that is heterologous to said promoter.
13. The isolated eukaryotic cell of claim 1 , wherein the heterologous polynucleotide further comprises an IRES.
14. The isolated eukaryotic cell of claim 13 , wherein the IRES is at least 95% identical to the sequence of SEQ ID NO: 1064.
15. The isolated eukaryotic cell of claim 1 , further comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1036.
16. The isolated eukaryotic cell of claim 1 , further comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 822.
17. The isolated eukaryotic cell of claim 1 , further comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 787.
18. The isolated eukaryotic cell of claim 1 , further comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 723.
19. The isolated eukaryotic cell of claim 1 , wherein the heterologous polynucleotide comprises an insulator.
20. The isolated eukaryotic cell of claim 19 wherein the insulator is at least 95% identical to the sequence of SEQ ID NO: 863.
21. The isolated eukaryotic cell of claim 1 , which is a mammalian cell.
22. The isolated eukaryotic cell of claim 4 , wherein the selective marker is a Sh ble gene.