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 - 20 . (canceled)
21 . A polynucleotide comprising a promoter operably linked to a heterologous nucleic acid to be expressed, and further comprising an intron at least 95% identical to a sequence of SEQ ID NO:964 or SEQ ID NO:966.
22 . The polynucleotide of claim 21 , wherein the promoter is selected from an EF1a promoter from any mammalian or avian species including human, rat, mice, chicken and Chinese hamster; a promoter from the immediate early genes 1, 2 and 3 of cytomegalovirus (CMV) from either human or murine cells; a promoter for eukaryotic elongation factor 2 (EEF2) from any mammalian or avian species including human, rat, mice, chicken and Chinese hamster; a GAPDH promoter from any mammalian or yeast species; an actin promoter from any mammalian or avian species including human, rat, mice, chicken and Chinese hamster; a PGK promoter from any mammalian or avian species including human, rat, mice, chicken and Chinese hamster; and a ubiquitin promoter.
23 . The polynucleotide of claim 21 , wherein the promoter is selected from sequences with SEQ ID NOS:892-941.
24 . The polynucleotide of claim 21 , further comprising a segment encoding a selectable marker.
25 . The polynucleotide of claim 24 , 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.
26 . The polynucleotide of claim 24 , wherein the amino acid sequence of the selectable marker comprises a sequence selected from SEQ ID NOS:701-703, 705-715 and 717-718.
27 . The polynucleotide of claim 24 , wherein the segment encoding the selectable marker is operably linked to a promoter comprising a sequence selected from SEQ ID NOS:937-948.
28 . polynucleotide of claim 24 , comprising a sequence selected from SEQ ID NOS:719-749 encoding the selectable marker and operably linked regulatory elements.
29 . The polynucleotide of claim 21 , further comprising an IRES.
30 . The polynucleotide of claim 29 , wherein the IRES is at least 95% identical to a sequence selected from SEQ ID NOS:1050-1094.
31 . The polynucleotide of claim 21 , further comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOS:820-858.
32 . The polynucleotide of claim 21 , further comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOS:751-819.
33 . The polynucleotide of claim 21 , wherein the polynucleotide further comprises an insulator.
34 . The polynucleotide of claim 33 , wherein the insulator is at least 95% identical to a sequence selected from one of SEQ ID NOS:859-865.
35 . The polynucleotide of claim 21 , further comprising a transposon end.
36 . The polynucleotide of claim 35 , wherein the transposon end comprises a sequence selected from SEQ ID NOS:1-38.
37 . A vector comprising the polynucleotide of claim 1 for expressing the heterologous nucleic acid in a mammalian cell.
38 . The vector of claim 37 , wherein the mammalian cell is a rodent cell.
39 . An isolated cell whose genome comprises the polynucleotide of claim 21 .