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 mammalian cell comprising:
 introducing into a mammalian cell.   a) a transposon comprising a heterologous polynucleotide flanked by a pair of transposon ends, wherein one transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO:25 and the other transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO:31, and wherein the heterologous polynucleotide comprises a promoter that is active in the mammalian cell; and   b) a  Bombyx  transposase comprising the amino acid sequence of SEQ ID NO:407;   wherein the transposase integrates the transposon into the genomic DNA of the mammalian cell.   
     
     
         21 . The method of  claim 20 , wherein one transposon end comprises a sequence that is at least 90% identical to SEQ ID NO: 25 and the other transposon end comprises a sequence that is at least 90% identical to SEQ ID NO: 31. 
     
     
         22 . The method of  claim 20 , wherein the heterologous polynucleotide comprises a promoter selected from an EF1a promoter, a CMV promoter, an EEF2 promoter, a GAPDH promoter, a Herpes Simplex Virus thymidine kinase (HSV-TK) promoter, an actin promoter, a PGK promoter, and an ubiquitin promoter. 
     
     
         23 . The method of  claim 20 , wherein the heterologous polynucleotide further comprises a second promoter, and wherein the transcription directions from the first and second promoters are different. 
     
     
         24 . The method of  claim 20 , wherein the promoter is operably linked to one or more of: i) an open reading frame; ii) a selectable marker; iii) a counter-selectable marker; iii) a nucleic acid encoding a regulatory protein; iv) a nucleic acid encoding an inhibitory RNA. 
     
     
         25 . The method of  claim 20 , wherein the heterologous polynucleotide comprises one or more sequence elements that increase expression by enhancing RNA processing or export from the nucleus. 
     
     
         26 . The method of  claim 25 , wherein the sequence elements are selected from WPRE, HPRE (SEQ ID NOS:104-105), SAR (SEQ ID NOS:108-111), AGS (SEQ ID NOS:106-107). 
     
     
         27 . The method of  claim 20 , wherein the heterologous polynucleotide comprises an insulator. 
     
     
         28 . The method of  claim 30 , wherein the insulator comprises a sequence that is at least 95% identical to a sequence selected from SEQ ID NOS:859-865. 
     
     
         29 . The method of  claim 20 , wherein the heterologous polynucleotide comprises a gene encoding an antibody chain or gene encoding a chimeric antigen receptor. 
     
     
         30 . The method of  claim 20 , wherein the transposase further comprises a heterologous nuclear localization signal (NLS) fused to the transposase. 
     
     
         31 . The method of  claim 20 , wherein the transposase is provided as a protein. 
     
     
         32 . The method of  claim 20 , wherein the transposase is provided as a nucleic acid encoding the transposase. 
     
     
         33 . The method of  claim 32 , wherein the nucleic acid is an mRNA molecule. 
     
     
         34 . The method of  claim 33 , wherein the mRNA molecule is expressed from a promoter operably linked to a gene encoding the transposase. 
     
     
         35 . The method of  claim 32 , wherein the nucleic acid further encodes a DNA binding domain (DBD) expressible as a fusion protein with the transposase. 
     
     
         36 . The method of  claim 35 , wherein the mammalian cell is a hamster cell, human cell or lymphocyte. 
     
     
         37 . The method of  claim 20 , further comprising identifying a cell with a stably integrated transposon. 
     
     
         38 . The method of  claim 37 , wherein the transposon of (a) comprises a gene encoding a selectable marker, and the identifying comprises growing the mammalian cell under conditions that provide a selective advantage to cells comprising said selectable marker. 
     
     
         39 . The method of  claim 38 , 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. 
     
     
         40 . The method of  claim 38 , 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. 
     
     
         41 . The method of  claim 38 , wherein the selectable marker comprises a gene encoding a fluorescent protein or a transmembrane protein. 
     
     
         42 . The method of  claim 38 , wherein the identifying comprises using flow cytometry. 
     
     
         43 . The method of  claim 20 , wherein the heterologous polynucleotide encodes a polypeptide, which is expressed in the mammalian cell.