Method for the modification of the expression characteristics of an endogenous gene of a given cell line

• US 5,272,071 A
• Filed: 05/28/1992
• Issued: 12/21/1993
• Est. Priority Date: 12/22/1989
• Status: Expired due to Term

• Current Assignee: Merck Serono SA (Merck & Co., Inc.)
• Original Assignee: Applied Research Systems ARS Holding NV (Merck & Co., Inc.)
• Inventors: Chappel, Scott C.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Escallon, Miguel
• Application Number:
  US07/893,447
• Time in Patent Office: 572 Days
• Field of Search: 435/172.3, 435/172.1, 435/69.1, 435/320.1, 435/252.3, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Normally transcriptionally silent genes in a cell line or microorganism may be activated for expression by inserting a DNA regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell or which is promiscuous, the regulatory element being inserted so as to be operatively linked with the normally silent gene in question. The insertion is accomplished by means of homologous recombination by creating a DNA construct including a segment having a DNA segment of the normally silent gene (targeting DNA) and the DNA regulatory element to induce gene transcription. The technique is also used to modify the expression characteristics of any endogenous gene of a given cell line or microorganism.

Claims

1. A method of activating a predetermined normally transcriptionally silent gene within the genome of a cell line so as to enable said cell line to express the gene product of said gene, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of stimulating expression of said gene when operatively linked thereto and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.

5. A method in accordance with claim 1, 2 wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.

6. A method in accordance with claim 1, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.

7. A method in accordance with claim 1, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.

8. A method in accordance with claim 1, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.

9. A method in accordance with claim 1, wherein said cell line is a eukaryotic cell line.

10. A method in accordance with claim 9, wherein said cell line is an animal cell line.

11. A method in accordance with claim 9, wherein said cell line is a mammalian cell line.

12. A method in accordance with claim 9, wherein said cell line is a plant cell line.

13. A method in accordance with claim 6, and additionally for causing expression of said gene product, further including the steps of, following said inserting step:

selecting clones of said cell line which express the product of said selectable marker gene;

cultivating the selected clones under conditions sufficient to permit expression of said gene product; and

collecting said gene product.

14. A method in accordance with claim 13, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.

15. A method in accordance with claim 13 wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.

16. A method in accordance with claim 15 wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.

2. A method of modifying, the expression characteristics of predetermined a gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of modifying the expression characteristics of said gene when operatively linked thereto, as compared to its existing DNA regulatory segment, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.

28. A method in accordance with claim 2, wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.

29. A method in accordance with claim 2, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.

30. A method in accordance with claim 2, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.

31. A method in accordance with claim 2, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.

32. A method in accordance with claim 2, wherein said cell line is a eukaryotic cell line.

33. A method in accordance with claim 32, wherein said cell line is an animal cell line.

34. A method in accordance with claim 32, wherein said cell line is a mammalian cell line.

35. A method in accordance with claim 32, wherein said cell line is a plant cell line.

36. A method in accordance with claim 29, and additionally for causing expression of said gene product, further including the steps of, following said inserting step:

selecting clones of said cell line which express the product of said selectable marker gene;

cultivating the selected clones under conditions sufficient to permit expression of said gene product; and

collecting said gene product.

37. A method in accordance with claim 36, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.

38. A method in accordance with claim 36, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.

39. A method in accordance with claim 38, wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.

3. A method of modifying the expression characteristics of a predetermined gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising an expressible, amplifiable gene capable of amplifying said gene when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said amplifiable gene is in sufficiently close proximity to said gene of interest to cause amplification thereof when said amplifiable gene is amplified.

4. A method in accordance with claim 3, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said expressible, amplifiable gene.

40. A method in accordance with claim 3, wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.

41. A method in accordance with claim 3, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.

42. A method in accordance with claim 3, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment to as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.

43. A method in accordance with claim 3, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.

44. A method in accordance with claim 3, wherein said cell line is a eukaryotic cell line.

45. A method in accordance with claim 44, wherein said cell line is an animal cell line.

46. A method in accordance with claim 44, wherein said cell line is a mammalian cell line.

47. A method in accordance with claim 44, wherein said cell line is a plant cell line.

48. A method in accordance with claim 41, and additionally for causing expression of said gene product, further including the steps of, following said inserting step:

selecting clones of said cell line which express the product of said selectable marker gene;

cultivating the selected clones under conditions sufficient to permit expression of said gene product; and

collecting said gene product.

49. A method in accordance with claim 48, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.

50. A method in accordance with claim 48, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.

51. A method in accordance with claim 50, wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.

17. A genome of a cell line, the genome having a DNA regulatory segment operatively linked with a predetermined naturally occurring gene at an insertion site characterized by a predetermined DNA sequence, said DNA regulatory segment not being naturally occurring at said location in the genome.

18. A cell line capable of expressing a gene product by a predetermined normally transcriptionally silent gene within the genome of said cell line, said genome having inserted therein a DNA regulatory segment operatively linked with said normally transcriptionally silent gene, said DNA regulatory segment being capable of promoting the expression of a gene product by said cell line.

22. A cell line in accordance with claim 18 wherein said DNA regulatory segment is one which is capable of promoting the expression of a gene product normally expressed by said cell line.

23. A cell line in accordance with claim 22, wherein the inserted DNA regulatory segment is part of a DNA construct comprising said DNA regulatory segment and at least one selectable marker gene.

24. A cell line in accordance with claim 23, wherein said DNA construct additionally comprises an amplifiable gene.

25. A method of obtaining a gene product from a cell line, comprising culturing a differentiated a cell line in accordance with claim 18 under conditions which permit expression of said gene product, and collecting said gene product.

56. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 22 under conditions which permit expression of said gene product, and collecting said gene product.

57. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 23 under conditions which permit expression of said gene product, and collecting said gene product.

58. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 24 under conditions which permit expression of said gene product, and collecting said gene product.

19. A cell line capable of enhanced expression of a gene product compared to the cell line from which it is derived, said gene product being the expression product of predetermined an endogenous gene within the genome of said cell, said genome having inserted therein in an operative manner, at or near said endogenous gene, an exogenous DNA regulatory segment and/or amplifiable gene capable of enhancing the expression of said gene product by said cell line.

20. A cell line in accordance with claim 19, wherein said exogenous DNA regulatory segment and/or amplifiable gene is an exogenous DNA regulatory segment.

21. A cell line in accordance with claim 19, wherein said exogenous DNA regulatory segment and/or amplifiable gene is an exogenous amplifiable gene.

52. A cell line in accordance with claim 20, wherein said DNA regulatory segment is one which is capable of promoting the expression of a gene product normally expressed by said cell line.

53. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 19 under conditions which permit expression of said gene product, and collecting said gene product.

54. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 20 under conditions which permit expression of said gene product, and collecting said gene product.

55. A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 21 under conditions which permit expression of said gene product, and collecting said gene product.

26. A DNA construct for insertion into a predetermined host cell line, comprising a DNA regulatory segment capable of modifying the expression characteristics of genes in the host cell line when operatively linked thereto and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.

27. A DNA construct for insertion into a predetermined host cell line, comprising an expressible, amplifiable gene capable of amplifying a gene in the host cell line when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.