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 non-naturally occurring transposase with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 407, wherein the transposase differs from the amino acid sequence of SEQ ID NO: 407 at least by one or more of the following amino acid substitutions (numbered according to SEQ ID NO: 407): Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E1785, E178H, C189P, A196G, L200I, L200M, A201Q, L211A, W215Y, T217K, G2195, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V323T, V324K, V324H, A330V, Q333M, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D4045, D404M, N441R, G448W, E449A, V469T, C473Q, R484K, T507C, G523A, I527M, Y528K, Y543I, E549A, K550M, P5575, V567I, V567H, E601V, E605H, E605W, D607H, 5609H or L610I, and the transposase has excision or transposition activity.
2. The polynucleotide of claim 1 , wherein the transposase also differs from the amino acid sequence of SEQ ID NO: 407 at least one of the following amino acid substitutions: Q85E, Q85M, Q85K, Q85H, Q85N, Q85T, Q85F, Q85L, Q92E, Q92P, Q92N, Q92I, Q92Y, Q92H, Q92F, Q92R, Q92D, Q92M, Q92W, Q92C, Q92G, Q92L, Q92V, Q92T, V93P, V93K, V93F, V93W, V93A, V93I, V93Q, P96A, P96T, P96M, P96R, P96V, P96E, P96Q, P96C, F97Q, F97K, F97T, F97W, F97V, F97E, F97P, F97D, F97A, F97R, F97G, F97N, F97Y, H165G, H165Q, H165T, H165M, H165V, H165L, H165C, H165N, H165D, H165K, H165A, E178Y, E178F, E178C, E178A, E178Q, E178G, E178V, E178D, E178L, E178P, E178W, C189D, C189Y, C189I, C189W, C189T, C189K, C189M, C189F, C189Q, C189V, L200I, L200F, L200C, L200Y, A201L, A201M, L203V, L203D, L203G, L203E, L203C, L203T, L203M, L203A, L203Y, N207G, N207A, L211G, L211M, L211C, L211T, L211V, T217V, T217A, T217I, T217P, T217C, T217Q, T217M, T217F, T217D, G219A, G219C, G219H, G219Q, Q235C, Q235N, Q235H, Q235W, Q235A, Q235T, Q235E, Q235M, Q235F, Q238C, Q238M, Q238H, Q238V, Q238T, Q238I, R242Q, N303C, N303G, N303A, N303D, N303S, N303H, N303E, N303R, N303K, N303L, N303Q, 1312F, 1312C, I312A, I312L, 1312T, 1312V, 1312G, 1312M, F321H, F321R, F321N, F321Y, F321G, F321E, F321M, F321K, F321A, F321Q, V323I, V323L, V323M, V323A, V324N, V324A, V324C, V324I, V324L, V324T, V324Y, V324F, V324S, V324Q, V324M, V324G, A330K, A330P, A330S, A330C, A330T, A330L, Q333P, Q333T, Q333H, Q333S, P337W, P337E, P337H, P337I, P337A, P337M, P337N, P337D, P337K, P337Q, P337G, P337S, P337C, P337L, P337V, F368Y, L373I, L373S, L373T, V389I, V389M, V389T, V389A, R394H, R394K, R394T, R394P, R394M, R394A, Q395P, Q395F, Q395E, Q395C, Q395V, Q395A, Q395H, Q395S, Q395Y, S399E, S399K, S399H, S399D, S399Y, S399G, S399Q, S399R, S399T, S399A, S399V, S399M, R402Y, R402D, R402F, R402G, R402N, R402E, R402M, R402S, R402Q, R402T, R402C, R402L, R402V, T403W, T403A, T403V, T403F, T403Y, T403N, T403G, T403C, T403I, T403S, T403M, T403Q, T403K, T403E, D404I, D404E, D404N, D404H, D404C, D404G, D404A, D404L, D404P, D404V, D404W, D404F, N408F, N408I, N408A, N408E, N408M, N408S, N408D, N408Y, N408H, N408C, N408Q, N408V, N408W, N408L, N408P, N408K, S409H, S409Y, S409N, S409I, S409D, S409F, S409T, S409C, S409Q, N441F, N441M, N441G, N441C, N441D, N441L, N441A, N441V, N441W, G448Y, G448H, G448C, G448T, G448V, G448N, G448Q, E449P, E449T, E449L, E449H, E449G, E449C, E449I, V469A, V469H, V469C, V469L, L472K, L472Q, L472M, C473G, C473T, C473I, C473M, R484H, T507R, T507D, T507S, T507G, T507K, T507I, T507M, T507E, T507L, T507V, G523Q, G523T, G523M, G523S, G523C, G523I, G523L, I527V, Y528N, Y528W, Y528M, Y528Q, Y528V, Y528I, Y528G, Y528D, Y528A, Y528E, Y528R, Y543C, Y543W, Y543M, Y543Q, Y543A, Y543R, Y543H, E549K, E549C, E549I, E549Q, E549H, E549C, E549M, E549S, E549F, E549L, K550R, K550Q, S556G, S556V, S556I, P557W, P557T, P557A, P557Q, P557K, P557D, P557G, P557N, P557L, P557V, H559K, H559S, H559C, H559I, H559W, V560F, V560P, V560I, V560H, V560Y, V560K, N561P, N561Q, N561G, N561A, V562Y, V562I, V562S, V562M, V567N, S583M, E601F, E601Q, E601W, E605R, E605K, E605M, E605P, E605Y, E605C, E605A, E605Q, E605S, E605V, E605I, E605G, D607V, D607Y, D607C, D607N, D607W, D607T, D607A, D607Q, D607E, D607L, D607K, D607G, S609R, S609W, S609V, S609Q, S609G, S609T, S609K, S609N, S609Y, L610T, L610K, L610G, L610A, L610W, L610D, L610Q, L610S, L610F, or L610N.
3. The polynucleotide of claim 1 , wherein the transposase comprises a sequence selected from one of the amino acid sequences of SEQ ID NO: 409-697.
4. The polynucleotide of claim 1 , wherein the transposase is fused to a heterologous nuclear localization sequence.
5. The polynucleotide of claim 1 , wherein the polynucleotide is an mRNA molecule.
6. The polynucleotide of claim 5 , wherein the mRNA comprises an anti-reverse cap analog (3′-O-Me-m 7 G(5′)ppp(5′)G.
7. The polynucleotide of claim 5 , wherein the mRNA is fully substituted with pseudo-uridine.
8. The polynucleotide of claim 5 , wherein the mRNA is fully substituted with 5-methyl-cytosine.
9. The polynucleotide of claim 1 , wherein the transposase comprises the amino acid sequence of SEQ ID NO: 415.
10. The polynucleotide of claim 1 , wherein a promoter is linked to the polynucleotide, wherein the polynucleotide is transcribable by a T7 RNA polymerase from the promoter, to produce an mRNA encoding the transposase.
11. The polynucleotide of claim 10 , wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 699.
12. The polynucleotide of claim 10 , further comprising the nucleotide sequence of SEQ ID NO: 700 3′ to the segment of the polynucleotide encoding the transposase.
13. The polynucleotide of claim 1 , wherein the transposase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 407.
14. The polynucleotide of claim 1 , wherein the transposase has at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 407.
15. The polynucleotide of claim 1 , wherein the polynucleotide comprises a promoter operably linked to the segment of the polynucleotide encoding the transposase.
16. A method of creating a transgenic cell, the method comprising introducing into a eukaryotic cell (a) the polynucleotide encoding the transposase of claim 15 , or a mRNA expressed therefrom, and (b) a polynucleotide comprising a transposon, wherein the transposon comprises transposon ends flanking a heterologous polynucleotide, wherein one of the transposon ends comprises at least 16 contiguous bases from the nucleic acid sequence of SEQ ID NO: 25 and the other of the transposon ends comprises at least 16 contiguous bases from the nucleic acid sequence of SEQ ID NO: 31; wherein the transposon is integrated into the genome of the eukaryotic cell thereby creating the transgenic cell.
17. The method of claim 16 , wherein the introducing is with the mRNA.
18. The method of claim 16 , wherein the transposon of (b) comprises a gene comprising a segment encoding a selectable marker wherein the segment is operably linked to a promoter, and the method further comprises identifying the transgenic cell by growing said eukaryotic cell under conditions that provide a selective advantage to cells expressing said selectable marker.
19. The method of claim 18 , wherein the promoter is at least 95% identical to a nucleotide sequence selected from one of the nucleotide sequences of SEQ ID NOS: 937-948.
20. The method of claim 18 , 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.