CA2507664A1 - Novel neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product - Google Patents
Novel neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product Download PDFInfo
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- CA2507664A1 CA2507664A1 CA002507664A CA2507664A CA2507664A1 CA 2507664 A1 CA2507664 A1 CA 2507664A1 CA 002507664 A CA002507664 A CA 002507664A CA 2507664 A CA2507664 A CA 2507664A CA 2507664 A1 CA2507664 A1 CA 2507664A1
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract 58
- 238000000034 method Methods 0.000 title claims 16
- 108010025815 Kanamycin Kinase Proteins 0.000 claims abstract 50
- 239000013604 expression vector Substances 0.000 claims abstract 18
- 210000004962 mammalian cell Anatomy 0.000 claims 47
- 210000004027 cell Anatomy 0.000 claims 17
- 150000001413 amino acids Chemical group 0.000 claims 16
- 235000001014 amino acid Nutrition 0.000 claims 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims 12
- 235000018102 proteins Nutrition 0.000 claims 11
- 102000004169 proteins and genes Human genes 0.000 claims 11
- 239000000047 product Substances 0.000 claims 8
- 239000003550 marker Substances 0.000 claims 7
- 239000004471 Glycine Substances 0.000 claims 6
- 239000003795 chemical substances by application Substances 0.000 claims 6
- 230000004544 DNA amplification Effects 0.000 claims 5
- 102000034287 fluorescent proteins Human genes 0.000 claims 5
- 108091006047 fluorescent proteins Proteins 0.000 claims 5
- 241000283984 Rodentia Species 0.000 claims 4
- 108010022394 Threonine synthase Proteins 0.000 claims 4
- 102000004419 dihydrofolate reductase Human genes 0.000 claims 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims 3
- 235000004279 alanine Nutrition 0.000 claims 3
- 239000012228 culture supernatant Substances 0.000 claims 3
- 239000003623 enhancer Substances 0.000 claims 3
- 238000000684 flow cytometry Methods 0.000 claims 3
- 102000004190 Enzymes Human genes 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 claims 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 claims 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims 2
- 101000849522 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 40S ribosomal protein S13 Proteins 0.000 claims 2
- 102000044159 Ubiquitin Human genes 0.000 claims 2
- 108090000848 Ubiquitin Proteins 0.000 claims 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims 2
- 229940024606 amino acid Drugs 0.000 claims 2
- 229960000485 methotrexate Drugs 0.000 claims 2
- 229920001184 polypeptide Polymers 0.000 claims 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims 2
- 239000004474 valine Substances 0.000 claims 2
- 241000699800 Cricetinae Species 0.000 claims 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims 1
- 235000003704 aspartic acid Nutrition 0.000 claims 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 229960000310 isoleucine Drugs 0.000 claims 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims 1
- 238000004114 suspension culture Methods 0.000 claims 1
- 230000035897 transcription Effects 0.000 claims 1
- 238000013518 transcription Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000010187 selection method Methods 0.000 abstract 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01095—Kanamycin kinase (2.7.1.95), i.e. neomycin-kanamycin phosphotransferase
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to novel modified neomycin phosphotransferase genes and the use thereof in a selection method for high-producing recombinant cells.
The invention also relates to expression vectors containing a modified neomycin phosphotransferase gene and a gene of interest in a functional bond with a heterologous promoter, and a method for producing heterologous gene products by using said expression vectors.
The invention also relates to expression vectors containing a modified neomycin phosphotransferase gene and a gene of interest in a functional bond with a heterologous promoter, and a method for producing heterologous gene products by using said expression vectors.
Claims (43)
1. Modified neomycin phosphotransferase gene characterised in that the modified neomycin phosphotransferase gene at amino acid position 91, 198 and/or 240 in relation to the wild-type gene codes for a different amino acid than the wild-type neomycin phosphotransferase gene.
2. Modified neomycin phosphotransferase gene according to claim 1, characterised in that the modified neomycin phosphotransferase which is encoded by the neomycin phosphotransferase gene has a lower enzyme activity than the wild-type neomycin phosphotransferase.
3. Modified neomycin phosphotransferase gene according to claim 1 or 2 characterised in that the modified neomycin phosphotransferase gene compared with the wild-type gene codes for alanine at amino acid position 91 in relation to the wild-type gene, for glycine at amino acid position 198, and/or for isoleucine at amino acid position 240.
4. Modified neomycin phosphotransferase gene according to one of claims 1 to 3 characterised in that the modified neomycin phosphotransferase gene codes for a polypeptide with an amino acid sequence according to SEQ
ID NO:6, SEQ SEQ ID NO:8 or SEQ ID NO:18.
ID NO:6, SEQ SEQ ID NO:8 or SEQ ID NO:18.
5. Modified neomycin phosphotransferase gene according to one of claims 1 to 4 containing or consisting of a sequence according to SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:17.
6. Modified neomycin phosphotransferase gene characterised in that the modified neomycin phosphotransferase gene compared with the wild-type gene codes for glycine at amino acid position 182 in relation to the wild-type gene, for alanine or valine at amino acid position 227 and/or for glycine at amino acid position 261.
7. Modified neomycin phosphotransferase gene according to claim 6 characterised in that the modified neomycin phosphotransferase gene codes for a polypeptide with an amino acid sequence according to SEQ
ID NO:4, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
ID NO:4, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
8. Modified neomycin phosphotransferase gene according to one of claims 6 or 7 containing or consisting of a sequence according to SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.
9. Modified neomycin phosphotransferase encoded by a modified neomycin phosphotransferase gene according to one of claims 1 to 8.
10. Eukaryotic expression vector containing a modified neomycin phosphotransferase gene according to one of claims 1 to 8.
11. Eukaryotic expression vector containing a heterologous gene of interest functionally linked to a heterologous promoter and a modified neomycin phosphotransferase gene which codes for a neomycin phosphotransferase having a lower enzyme activity compared with wild-type neomycin phosphotransferase.
12. Expression vector according to claim 11, characterised in that a) the modified neomycin phosphotransferase gene is a gene according to one of claims 1 to 5, or b) the modified neomycin phosphotransferase gene at amino acid position 182 and/or 227 codes for a different amino acid than the wild-type gene at the corresponding site, or c) the modified neomycin phosphotransferase gene at amino acid position 261 codes for a glycine.
13. Expression vector according to claim 12, characterised in that by comparison with the wild-type gene the modified neomycin phosphotransferase gene at amino acid position 182 codes for glycine or aspartic acid and/or by comparison with the wild-type gene at amino acid position 227 it codes for an alanine, glycine or valine.
14. Expression vector according to claim 12, characterised in that the modified neomycin phosphotransferase gene codes for a protein with an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID:20 or SEQ ID NO:22.
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID:20 or SEQ ID NO:22.
15. Expression vector according to one of claims 12 to 14, characterised in that it contains one or more enhancers functionally linked to the promoter or promoters.
16. Expression vector according to claim 15 characterised in that the enhancer is a CMV or SV40 enhancer.
17. Expression vector according to one of claims 11 to 16 characterised in that it contains a hamster ubiquitin/S27a promotor.
18. Expression vector according to claim 17 characterised in that the heterologous gene of interest is under the control of the ubiquitin/S27a promoter.
19. Expression vector according to one of claims 11 to 18 characterised in that it additionally contains a gene for a fluorescent protein which is/will optionally be functionally linked to the gene of interest and the heterologous promoter.
20. Expression vector according to claim 19, characterised in that it additionally contains an internal ribosome entry site (IRES) which enables bicistronic expression of the gene which codes for a fluorescent protein, and of the gene which codes for a protein/product of interest, under the control of a heterologous promoter.
21. Expression vector according to claim 19 or 20, characterised in that the gene which codes for a fluorescent protein, and the modified neomycin-phosphotransferase gene are located in one or in two separate transcription units.
22. Mammalian cell containing a modified neomycin phosphotransferase gene according to one of claims 1 to 8.
23. Mammalian cell which has been transfected with an expression vector according to one of Claims 10 to 18.
24. Mammalian cell which has been transfected with an expression vector according to one of Claims 19 to 21.
25. Mammalian cell according to one of claims 22 to 24, characterised in that it has additionally been transfected with a gene for an amplifiable selectable marker.
26. Mammalian cell according to claim 25, characterised in that the amplifiable selectable marker gene is dihydrofolate-reductase (DHFR).
27. Mammalian cell according to one of claims 22 to 26, characterised in that the mammalian cell is a rodent cell.
28. Mammalian cell according to claim 27, characterised in that the rodent cell is a CHO or BHK cell.
29. Method of enriching mammalian cells, characterised in that (i) a pool of mammalian cells is transfected with a gene for a modified neomycin-phosphotransferase according to one of claims 1 to 8 or with an E182D, D227G or D261N neomycin phosphotransferase mutant;
(ii) the mammalian cells are cultivated under conditions which allow expression of the modified neomycin-phosphotransferase gene; and (iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene.
(ii) the mammalian cells are cultivated under conditions which allow expression of the modified neomycin-phosphotransferase gene; and (iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene.
30. Method of obtaining and selecting mammalian cells which express at least one heterologous gene of interest, characterised in that (i) a pool of mammalian cells is transfected with at least one gene of interest and a gene for a modified neomycin-phosphotransferase according to one of claims 1 to 8 or with an E182D, D227G or D261N
neomycin phosphotransferase mutant;
(ii) the mammalian cells are cultivated under conditions which allow expression of the gene or genes of interest and of the modified neomycin-phosphotransferase gene; and (iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene.
neomycin phosphotransferase mutant;
(ii) the mammalian cells are cultivated under conditions which allow expression of the gene or genes of interest and of the modified neomycin-phosphotransferase gene; and (iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene.
31. Method according to claim 30, characterised in that the mammalian cells are additionally transfected with a gene for an amplifiable selectable marker and the selected mammalian cells are preferably subjected to at least one gene amplification step, the amplifiable selectable marker gene preferably being DHFR and the gene amplification preferably being carried out by the addition of methotrexate.
32. Method of obtaining and selecting mammalian cells which express at least one heterologous gene of interest, characterised in that (i) recombinant mammalian cells are transformed with an expression vector according to one of Claims 19 to 21;
(ii) are cultivated under conditions which allow expression of the gene (or genes) of interest, of the gene which codes for a fluorescent protein, and of the modified neomycin-phosphotransferase gene;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the mammalian cells are sorted by flow-cytometric analysis.
(ii) are cultivated under conditions which allow expression of the gene (or genes) of interest, of the gene which codes for a fluorescent protein, and of the modified neomycin-phosphotransferase gene;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the mammalian cells are sorted by flow-cytometric analysis.
33. Method according to claim 32, characterised in that the mammalian cells are additionally transfected with a gene for an amplifiable selectable marker and the cells sorted by flow-cytometric analysis are subjected to at least one gene amplification step, the amplifiable selectable marker gene preferably being DHFR and the gene amplification preferably being carried out by the addition of methotrexate.
34. Method of producing at least one protein of interest in recombinant mammalian cells, characterised in that (i) a pool of mammalian cells is transfected with at least one gene of interest and one gene for a modified neomycin-phosphotransferase according to one of claims 1 to 8 or with an E182D, D227G or D261N
neomycin phosphotransferase mutant;
(ii) the cells are cultivated under conditions which allow expression of the gene (or genes) of interest and of the modified neomycin-phosphotransferase;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the protein(s) of interest is or are obtained from the mammalian cells or the culture supernatant.
neomycin phosphotransferase mutant;
(ii) the cells are cultivated under conditions which allow expression of the gene (or genes) of interest and of the modified neomycin-phosphotransferase;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the protein(s) of interest is or are obtained from the mammalian cells or the culture supernatant.
35. Method of producing at least one protein of interest in recombinant mammalian cells, characterised in that (i) recombinant mammalian cells are transformed with an expression vector according to one of Claims 19 to 21;
(ii)are cultivated under conditions which allow expression of the gene (or genes) of interest, of the gene which codes for a fluorescent protein, and of the modified neomycin-phosphotransferase gene;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the mammalian cells are sorted by flow-cytometric analysis (v) the proteins) of interest is (are) obtained from the mammalian cells or the culture supernatant.
(ii)are cultivated under conditions which allow expression of the gene (or genes) of interest, of the gene which codes for a fluorescent protein, and of the modified neomycin-phosphotransferase gene;
(iii) the mammalian cells are cultivated in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene; and (iv) the mammalian cells are sorted by flow-cytometric analysis (v) the proteins) of interest is (are) obtained from the mammalian cells or the culture supernatant.
36. Method of producing at least one protein of interest, characterised in that (i) mammalian cells according to one of Claims 25 or 26 are cultivated under conditions which allow expression of the gene of interest, of the modified neomycin-phosphotransferase gene and of the amplifiable selectable marker gene;
(ii) the mammalian cells are cultivated and selected in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene;
(iii) the selected mammalian cells are subjected to at least one gene amplification step; and (iv) the protein(s) of interest is or are subsequently obtained from the mammalian cells or the culture supernatant.
(ii) the mammalian cells are cultivated and selected in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of those cells which express the modified neomycin-phosphotransferase gene;
(iii) the selected mammalian cells are subjected to at least one gene amplification step; and (iv) the protein(s) of interest is or are subsequently obtained from the mammalian cells or the culture supernatant.
37. Method according to one of Claims 34 to 36, characterised in that the mammalian cells are transfected with at least two genes of interest which code for a heteromeric protein/product, and (i) are cultivated under conditions which allow expression of the subunits of the heteromeric protein/product; and (ii) the heteromeric protein/product is isolated from the culture or culture medium.
38. Method according to one of Claims 34 to 37, characterised in that the sorted mammalian cells exhibit an average specific productivity of more than 5pg of the desired gene product (or products) per day and per cell.
39. Method according to claim 36 or 37, characterised in that the sorted host cells exhibit an average specific productivity of more than 20pg of the desired gene product (or products) per day and per cell.
40. Method according to one of Claims 29 to 39, characterised in that the mammalian cell is a rodent cell.
41. Method according to Claim 40, characterised in that the rodent cell is a CHO or BHK cell.
42. Method according to one of Claims 29 to 41, characterised in that the mammalian cells are cultivated in suspension culture.
43. Method according to one of Claims 29 to 42, characterised in that the mammalian cells are cultivated serum-free.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2706873A CA2706873C (en) | 2002-11-29 | 2003-11-25 | Modified neomycin-phosphotransferase gene that increases expression rates of co-integrated gene and uses thereof |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10256081.1 | 2002-11-29 | ||
| DE10256081 | 2002-11-29 | ||
| DE10330686.2 | 2003-07-08 | ||
| DE10330686A DE10330686A1 (en) | 2002-11-29 | 2003-07-08 | New neomycin phosphotransferase genes and methods for the selection of high-producing recombinant cells |
| PCT/EP2003/013203 WO2004050884A2 (en) | 2002-11-29 | 2003-11-25 | Novel neomycin phosphotransferase genes and method for the selection of high-producing recombinant cells |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2706873A Division CA2706873C (en) | 2002-11-29 | 2003-11-25 | Modified neomycin-phosphotransferase gene that increases expression rates of co-integrated gene and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2507664A1 true CA2507664A1 (en) | 2004-06-17 |
| CA2507664C CA2507664C (en) | 2010-09-07 |
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| CA2507664A Expired - Lifetime CA2507664C (en) | 2002-11-29 | 2003-11-25 | Novel neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP1567652B1 (en) |
| JP (1) | JP4344325B2 (en) |
| KR (1) | KR101149777B1 (en) |
| AT (1) | ATE371035T1 (en) |
| AU (1) | AU2003302689B2 (en) |
| BR (1) | BR0316525A (en) |
| CA (1) | CA2507664C (en) |
| DE (1) | DE50308030D1 (en) |
| DK (1) | DK1567652T3 (en) |
| ES (1) | ES2293096T3 (en) |
| MX (1) | MXPA05005481A (en) |
| WO (1) | WO2004050884A2 (en) |
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|---|---|---|---|---|
| US9045776B2 (en) | 2006-07-26 | 2015-06-02 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Regulatory nucleic acid elements |
| US9096878B2 (en) | 2008-12-22 | 2015-08-04 | National University Corporation Hokkaido University | Expression vector for producing protein derived from foreign gene in large quantity using animal cells, and use thereof |
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| EP1739179A1 (en) * | 2005-06-30 | 2007-01-03 | Octapharma AG | Serum-free stable transfection and production of recombinant human proteins in human cell lines |
| FR2897068B1 (en) * | 2006-02-03 | 2008-04-18 | Biomethodes Sa | IMPROVED KANAMYCINE KINASE II AND USES THEREOF |
| EP1975228A1 (en) * | 2007-03-28 | 2008-10-01 | Fachhochschule Mannheim | Polynucleotides for enhancing expression of a polynucleotide of interest |
| JP4491808B1 (en) * | 2009-06-05 | 2010-06-30 | 東洋紡績株式会社 | Drug selection marker gene expression cassette useful for efficient selection of high-expressing cells |
| JP4525863B1 (en) | 2009-06-05 | 2010-08-18 | 東洋紡績株式会社 | Expression vector for establishment of high-productivity cells and high-productivity cells |
| JP4547643B1 (en) * | 2009-06-05 | 2010-09-22 | 東洋紡績株式会社 | Expression vector optimized for cloning |
| US20120258496A1 (en) | 2010-09-27 | 2012-10-11 | Boehringer Ingelheim International Gmbh | Production of low fucose antibodies in h4-ii-e rat cells |
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| EP3536789A1 (en) | 2012-06-06 | 2019-09-11 | Boehringer Ingelheim International GmbH | Cell engineering using rnas |
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| EP2938726B1 (en) | 2012-12-31 | 2017-09-27 | Boehringer Ingelheim International GmbH | Heterologous intron within a signal peptide |
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| JPH04179485A (en) * | 1990-11-09 | 1992-06-26 | Junichi Miyazaki | New vector and method for selecting highly expressing transformed cell using the same vector |
| JP2004179485A (en) | 2002-11-28 | 2004-06-24 | Matsushita Electric Ind Co Ltd | Printed wiring board and method of manufacturing the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9045776B2 (en) | 2006-07-26 | 2015-06-02 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Regulatory nucleic acid elements |
| US9708626B2 (en) | 2006-07-26 | 2017-07-18 | Boehringer Ingelheim International Gmbh | Regulatory nucleic acid elements |
| US9096878B2 (en) | 2008-12-22 | 2015-08-04 | National University Corporation Hokkaido University | Expression vector for producing protein derived from foreign gene in large quantity using animal cells, and use thereof |
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| EP1567652B1 (en) | 2007-08-22 |
| JP2006507842A (en) | 2006-03-09 |
| DE50308030D1 (en) | 2007-10-04 |
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