Transposition of nucleic acids into eukaryotic genomes with a transposase from heliothis
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. A transposon comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 15 and the right end comprises SEQ ID NO: 16, wherein the transposon is transposable by a transposase having the sequence of SEQ ID NO:39.
2. The transposon of claim 1 , further comprising the sequence set forth by SEQ ID NO: 19 immediately adjacent to the left transposon end and between the left transposon end and the heterologous polynucleotide and the sequence set forth by SEQ ID NO: 22 immediately adjacent to the right transposon end and between the right transposon end and the heterologous polynucleotide.
3. The transposon of claim 1 , wherein the heterologous polynucleotide comprises a heterologous promoter that is active in eukaryotic cells.
4. The transposon of claim 3 , wherein the heterologous polynucleotide comprises an open reading frame in operable linkage with the heterologous promoter.
5. The transposon of claim 3 , wherein the heterologous promoter comprises a sequence selected from SEQ ID NOs: 723-812.
6. The transposon of claim 1 , wherein the heterologous polynucleotide comprises a heterologous enhancer that is active in eukaryotic cells.
7. The transposon of claim 6 , wherein the heterologous enhancer is selected from SEQ ID NOs: 702-722.
8. The transposon of claim 1 , wherein the heterologous polynucleotide comprises a heterologous intron that is spliceable in eukaryotic cells.
9. The transposon of claim 8 , wherein the nucleotide sequence of the heterologous intron is selected from SEQ ID NOs: 813-873.
10. The transposon of claim 1 , wherein the heterologous polynucleotide comprises an insulator sequence.
11. The transposon of claim 10 , wherein the nucleic acid sequence of the insulator sequence is selected from SEQ ID NOs: 684-690.
12. The transposon of claim 1 , wherein the heterologous polynucleotide comprises or encodes a selectable marker.
13. The transposon of claim 12 , wherein the selectable marker is selected from glutamine synthetase, dihydrofolate reductase, puromycin acetyltransferase, blasticidin acetyltransferase, hygromycin B phosphotransferase, aminoglycoside 3′-phosphotransferase—and a fluorescent protein.
14. A eukaryotic cell whose genome comprises a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 15 and the right end comprises SEQ ID NO: 16, wherein the transposon is transposable by a transposase having the sequence of SEQ ID NO:39.
15. The eukaryotic cell of claim 14 , wherein the cell is an animal cell.
16. The animal cell of claim 15 , wherein the animal cell is a mammalian cell.
17. The mammalian cell of claim 16 , wherein the mammalian cell is a rodent cell.
18. The mammalian cell of claim 16 , wherein the mammalian cell is a human cell.
19. The transposon of claim 1 , wherein the heterologous polynucleotide comprises two open reading frames, each operably linked to a separate promoter.
20. The transposon of claim 19 , wherein the heterologous polynucleotide further comprises a sequence selected from SEQ ID NOs: 997-1180.
21. The transposon of claim 3 , wherein the heterologous polynucleotide comprises in operable linkage with the heterologous promoter, one or more of: i) a nucleic acid encoding a selectable marker; ii) a nucleic acid encoding a counter-selectable marker; iii) a nucleic acid encoding a regulatory protein; and iv) a nucleic acid encoding an inhibitory RNA.
22. The transposon of claim 1 , wherein the target sequence 5′-TTAA-3′ is immediately adjacent to each of SEQ ID NOs:15 and 16 distal from the heterologous polynucleotide.
23. The eukaryotic cell of claim 14 , wherein the target sequence 5′-TTAA-3′ is immediately adjacent to each of SEQ ID NOs:15 and 16 distal from the heterologous polynucleotide.