Claims
- 1. An eukaryotic host cell genetically engineered to express a gene for a protein of interest and an IGF-1-signaling pathway gene.
- 2. The host cell of claim 1 wherein the IGF-1-signaling pathway gene is selected from the group consisting of a PKB gene, a MEK1 gene, a MEK2 gene, a glut5 gene, a glut1 gene, an ERK1 gene, an ERK2 gene, a JNK gene, a 14-3-3 protein gene, an IRS gene, and a P13 kinase gene.
- 3. The host cell of claim 2 wherein the IGF-1-signaling pathway gene is expressed under control of a heterologous regulatory element.
- 4. The host cell of claim 3, wherein the heterologous regulatory element is a viral promoter.
- 5. The host cell of claim 4, wherein the viral promoter is selected from the group consisting of a CMV promoter, an SV40 promoter, an RSV promoter and an adenoviral promoter.
- 6. The host cell of claim 1, wherein the protein of interest is selected from the group consisting of a soluble TNF receptor, a soluble IL-4 receptor, a soluble IL-1 type II receptor, a soluble Flt3 ligand, a soluble CD40 ligand, CD39, CD30, CD27, a TEK/Ork, IL-15, a soluble IL-15 receptor, Ox 40, GM-CSF, RANKL, RANK, TRAIL, a soluble TRAIL receptor, tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, an IL-2 receptor, an IL-2 antagonist, alpha-1 antitrypsin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, superoxide dismutase, glucagon, an erythropoeitin, an antibody, glucocerebrosidase, an Fc-fusion protein, globins, nerve growth factors, interleukins, colony stimulating factors, and immune response modifiers.
- 7. The host cell of claim 1, wherein the host cell is further genetically engineered to express a first selectable marker.
- 8. The host cell of claim 7, wherein the gene encoding the selectable marker is adjacent to the IGF-1-signaling pathway gene.
- 9. The host cell of claim 1, wherein the host cell is a mammalian cell.
- 10. The host cell of claim 9, wherein the host cell is selected from the group consisting of CHO, VERO, BHK, HeLa, CV1, MDCK, 293, 3T3, myeloma, PC12 and WI38 cells.
- 11. The host cell of claim 1, wherein the host cell is adapted to grow in serum-free medium.
- 12. The host cell of claim 11, wherein the host cell is a CHO cell and the IGF-1-signaling pathway gene is PKB.
- 13. The host cell of claim 11, wherein the host cell is a CHO cell and the IGF-1-signaling pathway gene is MEK.
- 14. The host cell of claim 1, wherein the host cell is genetically engineered to express a second IGF-1-signaling pathway gene.
- 15. The host cell of claim 14, wherein the IGF-1-signaling pathway gene is selected from the group consisting of PKB, MEK, ERK1 and ERK2.
- 16. A method of producing a protein of interest, the method comprising culturing an eukaryotic host cell genetically engineered to express a gene for a protein of interest and an IGF-1-signaling pathway gene under conditions such that the protein of interest is expressed.
- 17. The method of claim 16, wherein the IGF-1-signaling pathway gene is selected from the group consisting of a PKB gene, a MEK1 gene, a MEK2 gene, a glut5 gene, a glut1 gene, an ERK1 gene, an ERK2 gene, a JNK gene, a 14-3-3 protein gene, an IRS-1 gene, and a PI3 kinase gene.
- 18. The method of claim 16, further comprising collecting the protein of interest.
- 19. The method of claim 16, wherein the IGF-1-signaling pathway gene is expressed under control of a heterologous regulatory element.
- 20. The method of claim 19, wherein the heterologous regulatory element is a viral promoter.
- 21. The method of claim 20, wherein the viral promoter is selected from the group consisting of a CMV promoter, an SV40 promoter, an RSV promoter and an adenoviral promoter.
- 22. The method of claim 16, wherein the protein of interest is selected from the group consisting of a soluble TNF receptor, a soluble IL-4 receptor, a soluble IL-1 type II receptor, a soluble Flt3 ligand, a soluble CD40 ligand, CD39, CD30, CD27, a TEK/Ork, IL-1 5, a soluble IL-15 receptor, Ox 40, GM-CSF, RANKL, RANK, TRAIL, a soluble TRAIL receptor, tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, an IL-2 receptor, an IL-2 antagonist, alpha-1 antitrypsin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, superoxide dismutase, glucagon, an erythropoeitin, an antibody, glucocerebrosidase, an Fc-fusion protein, globins, nerve growth factors, interleukins, colony stimulating factors, and immune response modifiers.
- 23. The method of claim 16, wherein the host cell is further genetically engineered to express a first selectable marker.
- 24. The method of claim 23, wherein the gene encoding the selectable marker is adjacent to the IGF-1-signaling pathway gene.
- 25. The method of claim 16, wherein the host cell is a mammalian cell.
- 26. The method of claim 25, wherein the host cell is selected from the group consisting of CHO, VERO, BHK, HeLa, CV1, MDCK, 293, 3T3, myeloma, PC12 and W138 cells.
- 27. The method of claim 16, wherein the host cell is cultured in serum-free medium.
- 28. The method of claim 27, wherein the medium is growth-factor free.
- 29. The method of claim 27, wherein the medium is protein-free.
- 30. The method of claim 29, wherein the medium is peptone-free.
- 31. The method of claim 16, wherein the host cell is a CHO cell and the IGF-1-signaling pathway gene is PKB.
- 32. The method of claim 16, wherein the host cell is a CHO cell and the IGF-1-signaling pathway gene is MEK.
- 33. The method of claim 16, wherein the host cell is genetically engineered to express a second IGF-1-signaling pathway gene.
- 34. The method of claim 33, wherein the IGF-1-signaling pathway gene is selected from the group consisting of PKB, MEK, ERK1 and ERK2.
- 35. A method of producing an eukaryotic cell for production of a protein of interest, the method comprising genetically engineering an eukaryotic cell to express a gene that encodes a protein of interest, and to express an IGF-1-signaling pathway gene.
- 36. A method of producing a mammalian cell line capable of growth in serum-free medium, the method comprising exposing cells that have been genetically engineered to overexpress an IGF-1 signaling pathway gene to serum-free medium, and isolating a cell line that grows in serum-free medium.
- 37. The method of claim 36, further comprising exposing the cells to peptone-free medium, and isolating a cell line that grows in peptone-free medium.
- 38. The method of claim 36, further comprising exposing the cells to protein-free medium, and isolating a cell line that grows in protein-free medium.
- 39. The method of any one of claims 36 to 38, wherein the IGF-1 signaling pathway gene is a PKB gene.
- 40. The method of any one of claims 36 to 38, wherein the IGF-1 signaling pathway gene is a MEK gene.
- 41. The method of any one of claims 36 to 38, wherein the cell is genetically engineered to overexpress at least two IGF-1 signaling pathway genes.
- 42. The method of claim 41, wherein the IGF-1 signaling pathway genes are selected from the group consisting of a PKB gene, a MEK gene, and a MAPK gene.
- 43. A method of producing an eukaryotic cell for production of a protein of interest, the method comprising genetically engineering an eukaryotic cell to express a protein of interest, wherein the eukaryotic cell has been genetically engineered to express an IGF-1 signaling pathway gene.
- 44. A method of producing an eukaryotic cell for production of a protein of interest, the method comprising genetically engineering an eukaryotic cell to express an IGF-1 signaling pathway gene, wherein the eukaryotic cell expresses a protein of interest.
RELATED APPLICATIONS
[0001] The application is a divisional of U.S. patent application Ser. No. 09/648,667 filed Aug. 25, 2000, now allowed, and claims the benefit of U.S. Provisional Applications Serial No. 60/150,645 filed Aug. 25, 1999, Ser. No. 60/168,948 filed Dec. 3, 1999, and Ser. No. 60/171,949 filed Dec. 23, 1999, the disclosures of which are incorporated by reference in their entirety.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60150645 |
Aug 1999 |
US |
|
60168948 |
Dec 1999 |
US |
|
60171949 |
Dec 1999 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09648667 |
Aug 2000 |
US |
Child |
10109971 |
Mar 2002 |
US |