Dna vectors, transposons and transposases for eukaryotic genome modification

Abstract

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 - 19 . (canceled) 
     
     
         20 . A method of creating a transgenic cell comprising introducing into a eukaryotic cell a  Xenopus  transposon comprising the sequence 5′-CCYTTTBMCTGCCA-3′ (SEQ ID NO:19) inverted in orientation in the two transposon ends, and a  Xenopus  transposase, wherein the transposase integrates the transposon into the genome of the cell, wherein either
 i. the  Xenopus  transposase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 48 and which 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 a transposon from a polynucleotide of the nucleotide sequence of SEQ ID NO: 44 with increased activity relative to the transposase of the amino acid sequence of SEQ ID NO:48 fused to a heterologous nuclear localization signal, or 
 ii. the  Xenopus  transposase comprises the amino acid sequence of SEQ ID NO:48 or 49 fused to a heterologous nuclear localization sequence. 
 
     
     
         21 . The method of  claim 20  wherein the  Xenopus  transposase comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:48 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. 
     
     
         22 . The method of  claim 20  wherein the  Xenopus  transposase differs from the amino acid sequence of SEQ ID NO: 48 by at least one of the following amino acid substitutions: Y6C, S7G, M16S, S19G, S20Q, S20G, S20D, 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, K5771, I582R, Y583F, L587Y or L587W. 
     
     
         23 . The method of  claim 20 , wherein the  Xenopus  transposase comprises an amino acid sequence selected from one of SEQ ID NO: 51-406. 
     
     
         24 . The method of  claim 20 , wherein the  Xenopus  transposase is provided as a protein. 
     
     
         25 . The method of  claim 20 , wherein the  Xenopus  transposase is provided as a polynucleotide encoding the transposase. 
     
     
         26 . The method of  claim 25 , wherein the polynucleotide is an mRNA. 
     
     
         27 . The method of  claim 26 , wherein the mRNA is produced from a promoter operably linked to a gene encoding the transposase. 
     
     
         28 . The method of  claim 27 , wherein the mRNA is produced using a T7 polymerase. 
     
     
         29 . The method of  claim 28 , wherein the polynucleotide further comprises the nucleotide sequence of SEQ ID NO: 700 3′ to the segment of the polynucleotide encoding the transposase. 
     
     
         30 . The method of  claim 26 , wherein the mRNA comprises an anti-reverse cap analog (3′-O-Me-m7G(5′)ppp(5′)G. 
     
     
         31 . The method of  claim 26 , wherein the mRNA is fully substituted with pseudo-uridine. 
     
     
         32 . The method of  claim 26 , wherein the mRNA is fully substituted with 5-methyl-cytosine. 
     
     
         33 . The method of  claim 20 , wherein the transposase is fused to a DNA binding domain. 
     
     
         34 . The method of  claim 33 , wherein the DNA binding domain comprises a helix-turn-helix domain, a Zn-finger domain, a leucine zipper domain, or a helix-loop-helix domain. 
     
     
         35 . The method of  claim 20  further comprising identifying a cell in which the  Xenopus  transposon is incorporated into the genome of the eukaryotic cell. 
     
     
         36 . The method of  claim 35 , wherein the transposon comprises a gene encoding a selectable marker, and the identifying comprises growing the eukaryotic cell under conditions that provide a selective advantage to cells comprising the selectable marker. 
     
     
         37 . The method of  claim 36 , 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). 
     
     
         38 . The method of  claim 35 , wherein the transposon comprises a gene encoding a selectable marker, and the identifying comprises using flow cytometry. 
     
     
         39 . The method of  claim 28 , wherein the selectable marker comprises a gene encoding a fluorescent protein or a transmembrane protein. 
     
     
         40 . The method of  claim 20 , wherein one transposon end further comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO 9 and the other transposon end further comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO 17. 
     
     
         41 . The method of  claim 20 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         42 . The method of  claim 41 , wherein the mammalian cell is a hamster cell. 
     
     
         43 . The method of  claim 41 , wherein the mammalian cell is a human cell. 
     
     
         44 . The method of  claim 43 , wherein the human cell is a lymphocyte.