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 method of creating a transgenic mammalian cell comprising:
introducing into a mammalian cell
a) a Bombyx transposon comprising a first copy of a target site, a left transposon end, a heterologous polynucleotide, a right transposon end and a second copy of the target site, wherein the left transposon end comprises SEQ ID NO:24 and the right transposon end comprises SEQ ID NO:29, and the target site has the sequence 5′-TTAT-3′ or 5′-TTAA-3′, 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.
2. The method of claim 1 , 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.
3. The method of claim 1 , wherein the heterologous polynucleotide further comprises a second promoter, and wherein the transcription directions from the first and second promoters are different.
4. The method of claim 1 , wherein the promoter is operably linked to an open reading frame; or a nucleic acid encoding an inhibitory RNA.
5. The method of claim 1 , wherein the promoter is operably linked to a sequence encoding one of glutamine synthetase (GS), dihydrofolate reductase (DHFR), or a protein conferring resistance to puromycin, neomycin, hygromycin or zeocin.
6. The method of claim 1 , wherein the heterologous polynucleotide comprises one or more sequence elements that increase expression by enhancing RNA processing or export from the nucleus.
7. The method of claim 6 , wherein the sequence elements are selected from WPRE, HPRE (SEQ ID NOS:867-868), SAR (SEQ ID NO: 866), AGS (SEQ ID NOS:106-107).
8. The method of claim 1 , wherein the heterologous polynucleotide comprises an insulator.
9. The method of claim 8 , wherein the insulator comprises a sequence that is at least 95% identical to a sequence selected from SEQ ID NOS:859-865.
10. The method of claim 8 , wherein the heterologous polynucleotide comprises an internal ribosome entry site (IRES) sequence.
11. The method of claim 10 , wherein the IRES comprises a sequence selected from SEQ ID NOS: 1050-1094.
12. The method of claim 1 , wherein the heterologous polynucleotide comprises a gene encoding an antibody chain.
13. The method of claim 1 , wherein the heterologous polynucleotide comprises a gene encoding a chimeric antigen receptor.
14. The method of claim 1 , wherein the transposase further comprises a heterologous nuclear localization signal (NLS) fused to the transposase.
15. The method of claim 1 , wherein the transposase is provided as a protein.
16. The method of claim 1 , wherein the transposase is provided as a nucleic acid encoding the transposase.
17. The method of claim 16 , wherein the nucleic acid is an mRNA molecule.
18. The method of claim 17 , wherein the mRNA molecule is expressed from a promoter operably linked to a gene encoding the transposase.
19. The method of claim 17 , wherein the mRNA is expressed using a T7 polymerase.
20. The method of claim 17 , wherein the mRNA comprises the nucleotide sequence of SEQ ID NO:700 3′ to the segment of the polynucleotide encoding the transposase.
21. The method of claim 17 , wherein the mRNA comprises an anti-reverse cap analog (3′-O-Me-m7G(5′)ppp(5′)G.
22. The method of claim 17 , wherein the mRNA is fully substituted with pseudo-uridine.
23. The method of claim 17 , wherein the mRNA is fully substituted with 5-methyl-cytosine.
24. The method of claim 16 , wherein the nucleic acid further encodes a DNA binding domain (DBD) expressible as a fusion protein with the transposase.
25. The method of claim 1 , wherein the mammalian cell is a hamster cell.
26. The method of claim 1 , wherein the mammalian cell is a human cell.
27. The method of claim 26 , wherein the human cell is a lymphocyte.
28. The method of claim 1 , further comprising identifying a cell with a stably integrated transposon.
29. The method of claim 28 , wherein the transposon of (a) comprises a gene encoding a selectable marker, and the identifying comprises growing said mammalian cell under conditions that provide a selective advantage to cells comprising said selectable marker.
30. The method of claim 29 , 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.
31. The method of claim 29 , wherein the selectable marker is one of the following: glutamine synthetase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a protein that binds to zeocin.
32. The method of claim 29 , wherein the selectable marker comprises a gene encoding a fluorescent protein or a transmembrane protein.
33. The method of claim 29 , wherein the identifying comprises using flow cytometry.
34. The method of claim 1 , wherein the mammalian cell is a CHO cell or a human cell.