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 - 20 . (canceled) 
     
     
         21 . A polynucleotide comprising a left piggybac-like transposon end and a right piggybac-like transposon end flanking a heterologous polynucleotide comprising a mouse CMV promoter operably linked to a heterologous nucleic acid sequence to be expressed. 
     
     
         22 . The polynucleotide of  claim 21 , wherein the left piggybac-like transposon end is at least 90% identical to SEQ ID NO: 35 and the right piggybac-like transposon end is at least 90% identical to SEQ ID NO: 36. 
     
     
         23 . The polynucleotide of  claim 21 , wherein the mouse CMV promoter is selected from SEQ ID NOS: 916-920. 
     
     
         24 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises an intron comprising a sequence 95% identical to a sequence selected from SEQ ID NOS: 958-997. 
     
     
         25 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprising an enhancer. 
     
     
         26 . The polynucleotide of  claim 21 , wherein the enhancer is at least 95% identical to a sequence selected from SEQ ID NO: 877-891. 
     
     
         27 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises a segment encoding a selectable marker. 
     
     
         28 . The polynucleotide of  claim 27 , 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. 
     
     
         29 . The polynucleotide of  claim 27 , wherein the segment encoding the selectable marker is operably linked to a promoter comprising a sequence selected from SEQ ID NOS: 937-948. 
     
     
         30 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises a second promoter. 
     
     
         31 . The polynucleotide of  claim 30 , wherein the promoter is selected from one of the following: an EF1a promoter, a human and mouse CMV promoter, an EEF2 promoter, an SV40 promoter, a PGK promoter, an actin promoter, a GAPDH promoter, an ILV5 promoter or an HSV-TK promoter. 
     
     
         32 . The polynucleotide of  claim 31 , wherein the promoter comprises a sequence selected from SEQ ID NO: 892-936. 
     
     
         33 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 998-1049. 
     
     
         34 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 751-819. 
     
     
         35 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 719-749. 
     
     
         36 . The polynucleotide of  claim 21 , wherein the heterologous polynucleotide further comprises an insulator. 
     
     
         37 . The polynucleotide of  claim 36 , wherein the insulator is at least 95% identical to a sequence selected from one of SEQ ID NOS: 859-865. 
     
     
         38 . A vector comprising the polynucleotide of  claim 21 , wherein the heterologous nucleic acid sequence can be expressed in a mammalian cell. 
     
     
         39 . The vector of  claim 38 , wherein the mammalian cell is a rodent cell. 
     
     
         40 . An isolated cell whose genome comprises the polynucleotide of  claim 21 .