Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials

• US 4,399,216 A
• Filed: 02/25/1980
• Issued: 08/16/1983
• Est. Priority Date: 02/25/1980
• Status: Expired due to Term

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Axel, Richard, Wigler, Michael H., Silverstein, Saul J.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/124,513
• Time in Patent Office: 1,268 Days
• Field of Search: 435/68, 435/172, 435/70, 435/240, 435/241, 435/948, 435/811, 435/6, 435/29, 435/34, 424/85, 424/177, 424/178, 424/180
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: Y10S 435/811 Interferon
• CPC Class Codes: Y10S 435/948 using viruses or cell lines

Abstract

The present invention relates to processes for inserting DNA into eucaryotic cells, particularly DNA which includes a gene or genes coding for desired proteinaceous materials for which no selective criteria exist. The insertion of such DNA molecules is accomplished by cotransforming eucaryotic cells with such DNA together with a second DNA which corresponds to a gene coding for a selectable marker.

The invention further relates to processes for inserting into eucaryotic cells a multiplicity of DNA molecules including genes coding for desired proteinaceous materials by cotransformation with the desired genes and with amplifiable genes for a dominant selectable marker in the presence of successively higher amounts of an inhibitor. Alternatively, the insertion of multiple copies of desired genes is accomplished by transformation using DNA molecules formed by ligating a DNA molecule including the desired gene to a DNA molecule which includes an amplifiable gene coding for a dominant selectable phenotype such as a gene associated with resistance to a drug in the presence of successively higher amounts of an agent such as a drug against which the gene confers resistance so that only those eucaryotic cells into which multiple copies of the amplifiable gene have been inserted survive.

Claims

1. A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said ducaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.

2. A process in accordance with claim 1 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

3. A process in accordance with claim 2 wherein said foreign DNA I codes for interferon protein.

4. A process in accordance with claim 2 wherein said foreign DNA I codes for insulin.

5. A process in accordance with claim 2 wherein said foreign DNA I codes for growth hormone.

6. A process in accordance with claim 2 wherein said foreign DNA I codes for a clotting factor.

7. A process in accordance with claim 2 wherein said foreign DNA I codes for a viral antigen or an antibody.

8. A process in accordance with claim 2 wherein said foreign DNA I codes for an enzyme.

9. A process in accordance with claim 1 wherein said foreign DNA I is substantially purified.

10. A process in accordance with claim 1 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

11. A process in accordance with claim 1 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

12. A process in accordance with claim 1 wherein said eucaryotic cell is a mammalian cell.

13. A process in accordance with claim 12 wherein said mammalian cell is an erythroblast.

14. A process in accordance with claim 12 wherein said mammalian cell is a fibroblast.

15. A process in accordance with claim 1 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for a selectable phenotype in the range from about 1:

1 to about 100,000;

1.

16. A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises the gene for thymidine kinase from herpes simplex virus.

17. A process in accordance with claim 1 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises the gene for adenine phosphoribosyltransferase.

18. A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises a gene associated with drug resistance.

19. A process in accordance with claim 18 wherein said gene associated with drug resistance is the gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

20. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 1.

21. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 1.

22. A process for producing a foreign proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 1, culturing or cloning said cotransformed eucaryotic cell under suitable conditions to yield a multiplicity of eucaryotic cells producing said foreign proteninaceous material and recovering said proteinaceous material from said eucaryotic cells.

23. A process in accordance with claim 22 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

24. A process in accordance with claim 22 wherein said eucaryotic cell is a mammalian cell.

25. A method of detecting eucaryotic cells which have been transformed with foreign DNA I which is not associated with a selectable phenotype which comprises cotransforming said eucaryotic cell with said DNA I and with DNA II which is associated with a selectable phenotype in accordance with the process of claim 1, and screening for eucaryotic cells so cotransformed.

26. A process for inserting foreign DNA I into a eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out in a suitable medium and in the presence of conditions permitting identification and recovery of eucaryotic cells which have acquired said selectable phenotype.

27. A process for cotransforming a suitable eucaryotic cell which comprises transforming under suitable conditions said eucaryotic cell with foreign DNA I and with foreign DNA II, said DNA I and DNA II being unlinked and said DNA II coding for a selectable phenotype not expressed by said eucaryotic cell prior to cotransformation.

28. A process for inserting purified foreign DNA I coding for proteinaceous material which is not associated with a selectable phenotype into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II coding for proteinaceous material which is associated with a selectable phenotype, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.

29. A process in accordance with claim 28 wherein said proteinaceous material which is not associated with a selectable phenotype comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

30. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 28.

31. A process for inserting a multiplicity of foreign DNA I molecules corresponding to multiple copies of a gene coding for a proteinaceous material into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said multiplicity of foreign DNA I molecules and with a multiplicity of unlinked foreign DNA II molecules coding for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said multiplicity of genes coding for said selectable phenotype.

32. A process in accordance with claim 31 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

33. A process in accordance with claim 32 wherein said foreign DNA I codes for interferon protein.

34. A process in accordance with claim 32 wherein said foreign DNA I codes for insulin.

35. A process in accordance with claim 32 wherein said foreign DNA I codes for growth hormone.

36. A process in accordance with claim 32 wherein said foreign DNA I codes for a clotting factor.

37. A process in accordance with claim 32 wherein said foreign DNA I codes for a viral antigen or an antibody.

38. A process in accordance with claim 32 wherein said foreign DNA I codes for an enzyme.

39. A process in accordance with claim 31 wherein said foreign DNA I is substantially purified.

40. A process in accordance with claim 31 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

41. A process in accordance with claim 31 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

42. A process in accordance with claim 31 wherein said eucaryotic cell is a mammalian cell.

43. A process in accordance with claim 42 wherein said mammalian cell is an erythroblast.

44. A process in accordance with claim 42 wherein said mammalian cell is a fibroblast.

45. A process in accordance with claim 31 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:

1 to about 100,000;

1.

46. A process in accordance with claim 31 wherein said foreign DNA II which does for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with drug resistance.

47. A process in accordance with claim 46 wherein said gene associated with drug resistance is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

48. A process in accordance with claim 31 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.

49. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 31.

50. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 31.

51. A process for producing a foregin proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 31, maintaining said cotransformed eucaryotic cell under suitable conditions to produce said foreign proteinaceous material, and recovering said proteinaceous material so produced.

52. A process in accordance with claim 51 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

53. A process in accordance with claim 51 wherein said eucaryotic cell is mammalian cell.

54. A process for generating a multiplicity of foregin DNA I molecules corresponding to multiple copies of a gene in a eucaryotic cell which comprises tranforming said eucaryotic cell with a molecule which is formed by linking one of said foreign DNA I molecules to a DNA II molecule corresponding to an amplifiable gene for a dominant selectable phenotype not expressed by said eucaryotic cell, and culturing the transformed eucaryotic cells in the presence of successively elevated concentrations of an agent permitting survival or identification of eucaryotic cells which have acquired multiple copies of said amplifiable gene, said transformation and culturing being carried out under suitable conditions.

55. A process in accordance with claim 54 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

56. A process in accordance with claim 55 wherein said foreign DNA I codes for interferon protein.

57. A process in accordance with claim 55 wherein said foreign DNA I codes for insulin.

58. A process in accordance with claim 55 wherein said foreign DNA I codes for growth hormone.

59. A process in accordance with claim 55 wherein said foreign DNA I codes for a clotting factor.

60. A process in accordance with claim 55 wherein said foreign DNA I codes for a viral antigen or antibody.

61. A process in accordance with claim 55 wherein said foregin DNA I codes for an enzyme.

62. A process in accordance with claim 54 wherein said foreign DNA I is substantially purified.

63. A process in accordance with claim 54 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

64. A process in accordance with claim 54 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

65. A process in accordance with claim 54 wherein said eucaryotic cell is mammalian cell.

66. A process in accordance with claim 65 wherein said mammalian cell is an erythroblast.

67. A process in accordance with claim 65 wherein said mammalian cell is a fibroblast.

68. A process in accordance with claim 54 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:

1 to about 100,000;

1.

69. A process in accordance with claim 54 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with resistance to a drug or chemical antagonist.

70. A process in accordance with claim 69 wherein said gene associated with resistance to a drug or chemical anatgonist is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

71. A process in accordance with claim 54 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.

72. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 54.

73. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 54.