Immortalization and disimmortalization of cells

• US 5,629,159 A
• Filed: 06/07/1995
• Issued: 05/13/1997
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Anderson, David J.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US08/481,127
• Time in Patent Office: 706 Days
• Field of Search: 435/240.2, 435/172.3, 435/320.1, 435/6, 435/69.1, 435/194
• US Class Current: 435/6.16
• CPC Class Codes: A61K 38/162 from virus
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2740/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

Methods and compositions for the conditional immortalization of cells are provided.

Claims

1. An immortalized mammalian cell that contains in the genome of said cell exogenous nucleic acid comprising at least a first and a second recombinase target site flanking an immortalization gene, wherein said first and second target sites mediate excision of said immortalization gene when said first and second target sites are contacted with a recombinase.

2. An immortalized cell line according to claim 1 wherein said exogeneous nucleic acid further comprises a selection marker gene.

3. An immortalized cell line according to claim 2 wherein said selection marker gene is a negative selection marker gene.

4. An immortalized cell line according to claim 2 wherein said nucleic acid further comprises a STOP site.

5. An immortalized cell line according to claim 1 wherein said exogeneous nucleic acid further comprises a first selection marker gene and a second selection marker gene.

6. An immortalized cell line according to claim 5 wherein said second selection marker gene is not between the first and second recombinase target sites.

28. A cell according to claim 1 wherein said recombinase is Cre recombinase.

34. A cell according to claim 1 wherein said recombinase is FLP recombinase.

7. A method for creating an immortalized cell that contains recombinase target sites flanking an immortalization gene in the genome of said immortalized cell, said method comprising transforming a cell with exogeneous nucleic acid comprising

a) a first recombinase site;

b) an immortalization gene; and

c) a second recombinase site;

such that in the absence of a recombinase said exogeneous nucleic acid is incorporated into the genome of said cell.

8. A method according to claim 7 wherein said nucleic acid further comprises at least one selection marker gene.

9. A method according to claim 8 wherein said selection marker gene is a negative selection marker.

29. A method according to claim 7 wherein said recombinase is Cre recombinase.

35. A method according to claim 7 wherein said recombinase is FLP recombinase.

10. A method for the disimmortalization of an immortalized cell that contains exogeneous nucleic acid comprising recombinase target sites flanking an immortalization gene in the genome of said immortalized cell, said method comprising contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites, wherein said recombinase is produced by expression of a recombinase nucleic acid within said cell.

11. A method according to claim 10 wherein said expression is achieved by transforming said immortalized cell with a recombinase nucleic acid encoding said recombinase and maintaining said transformed cells under conditions where said recombinase is expressed and said immortalization gene is excised.

30. A method according to claim 10 wherein said recombinase is Cre recombinase.

36. A method according to claim 10 wherein said recombinase is FLP recombinase.

12. A method for the disimmortalization of an immortalized cell comprising:

a) incorporating exogeneous nucleic acid comprising;

i) a first recombinase target site;

ii) an immortalization gene;

iii) a negative selection marker gene; and

iv) a second recombinase target site;

into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene and the negative selection marker; and

b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene and said negative selection marker gene are excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.

13. A method according to claim 12 wherein said expression is achieved by transforming said immortalized cells with a vector encoding a recombinase which recognizes said recombinase target sites under conditions wherein said recombinase is expressed.

14. A method according to claim 12 wherein said exogeneous nucleic acid further comprises a gene for a recombinase that recognizes said recombinase target sites, wherein said recombinase gene is operably linked to an inducible promoter, and said expression is achieved by growing said immortalized cells under conditions wherein said recombinase is expressed.

15. A method according to claim 12 wherein said exogeneous nucleic acid further comprises a gene for a recombinase that recognizes said recombinase target sites, wherein said recombinase gene is operably linked to an inducible promoter, and said contacting is achieved by growing said immortalized cells under conditions wherein said recombinase is expressed.

16. A method according to claim 12 wherein said method further comprises selecting for disimmortalized cells in the presence of a negative selection agent.

17. A method according to claim 12 wherein said exogeneous nucleic acid further comprises a positive selection gene.

20. A method according to claim 17 wherein said expression is achieved by transforming said immortalized cells with a vector encoding a recombinase which recognizes said recombinase target sites under conditions wherein said recombinase is expressed.

21. A method according to claim 17 wherein said exogeneous nucleic acid further comprises a gene for a recombinase that recognizes said recombinase target sites operably linked to an inducible promoter, and said expression is achieved by growing said immortalized cells under conditions wherein said recombinase is expressed.

31. A method according to claim 12 wherein said recombinase is Cre recombinase.

37. A method according to claim 12 wherein said recombinase is FLP recombinase.

18. A method for the disimmortalization of an immortalized cell comprising:

a) incorporating exogeneous nucleic acid comprising;

i) a first recombinase target site;

ii) an immortalization gene;

iii) a selection marker gene; and

iv) a second recombinase target site;

into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene, resulting in the expression of the selection marker; and

b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.

19. A method according to claim 18 wherein said nucleic acid further comprises a STOP site.

22. A method according to claim 18 wherein said method further comprises selecting for disimmortalized cells that express said selection marker gene.

32. A method according to claim 18 wherein said recombinase is Cre recombinase.

38. A method according to claim 18 wherein said recombinase is FLP recombinase.

23. A method for the disimmortalization of an immortalized cell comprising:

a) incorporating exogeneous nucleic acid comprising;

i) a first recombinase target site;

ii) an immortalization gene;

iii) a first selection marker gene;

iv) a second recombinase target site; and

v) a second selection marker gene;

into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that when the immortalization gene is expressed, the first selection marker gene is also expressed and the second selection marker gene is not expressed and when said immortalization gene is excised said second selection marker is expressed; and

b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.

24. A method according to claim 23 wherein said expression is by transforming said immortalized cells with a vector encoding a recombinase which recognizes said recombinase target sites under conditions wherein said recombinase is expressed.

25. A method according to claim 23 wherein said exogeneous nucleic acid further comprises a gene for a recombinase that recognizes said recombinase target sites, wherein said recombinase gene is operably linked to an inducible promoter, and said expression is by growing said immortalized cells under conditions wherein said recombinase is expressed.

26. A method according to claim 23 wherein said nucleic acid further comprises an additional exogeneous gene.

27. A method according to claim 23 wherein said nucleic acid further comprises a STOP site.

33. A method according to claim 23 wherein said recombinase is Cre recombinase.

39. A method according to claim 23 wherein said recombinase is FLP recombinase.