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. A polynucleotide encoding a transposase, other than a naturally occurring protein, wherein the transposase is at least 90% identical to SEQ ID NO: 61 and differs from SEQ ID NO:48 by at least one amino acid shown in Table 4 column C or D, and wherein the transposase, when fused to a heterologous nuclear localization signal, can excise or transpose a transposon from SEQ ID NO: 44 with at least two-fold increased excision or transposition activity respectively relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
2. The polynucleotide of claim 1 , wherein the transposase can transpose a transposon from SEQ ID NO: 44, by excising the transposon and integrating it into the genomic DNA of a target cell with at least 2-fold increased transposition activity relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
3. The polynucleotide of claim 1 , wherein the transposase encoded by the polynucleotide comprises at least one amino acid substitution at one of the following positions (numbered according to SEQ ID NO: 48): 6, 7, 16, 19, 20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141, 145, 146, 148, 150, 157, 162, 179, 182, 189, 192, 193, 196, 198, 200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336, 354, 357, 358, 359, 377, 423, 426, 428, 438, 447, 447, 450, 462, 469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583 or 587.
4. The polynucleotide of claim 1 , wherein the transposase differs from SEQ ID NO: 48 by at least one of the following amino acid substitutions: Y6C, S7G, M16S, S19G, 520Q, 520G, 520D, E21D, E22Q, F23T, F23P, S24Y, S26V, S28Q, V31K, A34E, L67A, G73H, A76V, D77N, P88A, N91D, Y141Q, Y141A, N145E, N145V, P146T, P146V, P146K, P148T, P148H, Y150G, Y150S, Y150C, H157Y, A162C, A179K, L182I, L182V, T189G, L192H, S193N, S193K, V196I, S198G, T200W, L210H, F212N, N218D, N218E, A248N, L263M, Q270L, S294T, T297M, 5308R, L310R, L333M, Q336M, A354H, C357V, L358F, D359N, L377I, V423H, P426K, K428R, S438A, T447G, T447A, L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E517I, P520D, P520G, N523S, I533E, D534A, F576R, F576E, K577I, I582R, Y583F, L587Y or L587W.
5. The polynucleotide of claim 1 , wherein the transposase comprises a sequence selected from one of SEQ ID NO: 51-406.
6. The polynucleotide of claim 1 , wherein the polynucleotide is an mRNA molecule.
7. The polynucleotide of claim 6 , wherein the mRNA comprises an anti-reverse cap analog (3′-O-Me-m 7 G(5′)ppp(5′)G.
8. The polynucleotide of claim 6 wherein the mRNA is fully substituted with pseudo-uridine.
9. The polynucleotide of claim 6 wherein the mRNA is fully substituted with 5-methyl-cytosine.
10. The polynucleotide of claim 1 , wherein the polynucleotide comprises a promoter operably linked to a segment of the polynucleotide encoding the transposase.
11. The polynucleotide of claim 10 , wherein the polynucleotide is transcribable by a T7 RNA polymerase from the promoter to mRNA encoding the transposase.
12. The polynucleotide of claim 11 , wherein the promoter comprises SEQ ID NO: 699.
13. The polynucleotide of claim 11 further comprising SEQ ID NO: 700 3′ to the segment of the polynucleotide encoding the transposase.
14. A method of creating a transgenic cell comprising (i) introducing into a eukaryotic cell (a) the transposase of claim 1 and (b) a polynucleotide comprising a transposon comprising transposon ends of a piggyBac-like transposon flanking a heterologous polynucleotide, wherein one transposon end comprises at least 16 contiguous bases from SEQ ID NO: 7 and the other transposon end comprises at least 16 contiguous bases from SEQ ID NO: 16 and (ii) identifying a cell in which the transposon is incorporated into the genome of the eukaryotic cell.
15. The method of claim 14 , wherein the transposase is provided as a protein.
16. The method of claim 14 , wherein the transposase is provided as a polynucleotide encoding the transposase.
17. The method of claim 16 , wherein the polynucleotide is an mRNA.
18. The method of claim 16 , wherein the polynucleotide comprises a promoter operably linked to a segment of the polynucleotide encoding the transposase.
19. The method of claim 14 , wherein the transposon of (i)(b) comprises a gene encoding a selectable marker, and the identifying of (ii) comprises growing the eukaryotic cell under conditions that provide a selective advantage to cells comprising the selectable marker.
20. The method of claim 19 , wherein the gene encoding the selectable marker is operably linked to a promoter which is at least 95% identical to a sequence selected from SEQ ID Nos: 937-948.
21. The method of claim 19 , 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 (eg the Sh ble gene).
22. A polynucleotide encoding a transposase linked to a heterologous nuclear localization sequence, wherein the transposase is other than a naturally occurring protein, at least 90% identical to SEQ ID NO: 61, and differs from SEQ ID NO:48 by at least one amino acid shown in Table 4 column C or D and can excise a transposon from SEQ ID NO: 44 with increased activity relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.